The objective was to explore the pharmacological mechanism of modified shengmaiyin (MSMY) in the treatment of acute lymphoblastic leukemia (ALL) by network pharmacology analysis. The effective components and predicted targets of MSMY were collected from TCMSP and Swiss target prediction databases, and the related targets of ALL were screened by GeneCards and DisGeNET. The core targets and related signaling pathways of MSMY active ingredients for the treatment of ALL were predicted by protein-protein interaction network (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. We identified 172 potential targets for the active components of MSMY, 538 disease targets associated with ALL, and 59 common gene targets. PPI network showed that 27 targets such as triptolide, RAC-alpha serine/threonine-protein kinase (AKT1), vascular endothelial growth factor A and Caspase-3 (CASP3) were the core targets. KEGG enrichment analysis related signaling pathways included cancer pathway, phosphatidylinositol 3 kinase, PI-3K/protein kinase B (PI3K-Akt) signaling pathway, apoptosis and mitogen-activated protein kinase (MAPK) signaling pathway and IL-17 signaling pathway. The effective active components and potential therapeutic targets of MSMY in the treatment of ALL were initially identified by comprehensive network pharmacology, which provides a theoretical basis for further study of the material basis and molecular mechanism of MSMY in the treatment of ALL. At present, chemotherapy is the first recommended treatment for ALL. With the progress of modern chemotherapy technology, the outcome of ALL patients has been significantly improved, and the 5-year overall survival rate has been significantly improved. The 5-year overall survival rate of children with ALL can reach 90%. However, the adverse reactions caused by chemotherapy drugs, such as gastrointestinal reactions caused by chemotherapy and affecting the metabolism of nutrients in the body, can cause malnutrition. The bone marrow suppression caused by ALL chemotherapy can lead to the decline of immune function, secondary infection, bleeding, etc, so that children cannot tolerate chemotherapy, reduce the quality of life, and even have to interrupt chemotherapy, leading to treatment failure, and the related side effects seriously limit the clinical use of chemotherapy. Traditional Chinese Medicine (TCM) classifies ALL as consumptive disease, blood syndrome, malignant nucleus and so on according to different clinical manifestations, and holds that its pathogenesis is warm toxin, pathogenic toxin attacking and touching, pathogenic toxin burning nutrient yin, torturing viscera essence and blood, and damaging bone marrow. At the same time, studies have found that TCM treatment of ALL plays a greater role in inhibiting proliferation, promoting apoptosis, inducing differentiation, reversing drug resistance, enhancing efficacy and attenuating toxicity, and regulating immune function. Moreover, TCM has been widely recognized in prolonging the survival time of patients with ALL, reducing adverse reactions, enhancing therapeutic effect and reversing drug resistance.Acute lymphoblastic leukemia (ALL) refers to the abnormal differentiation of lymphocytes in the early stage, resulting in malignant proliferation and abnormal blood function. The pathological manifestation of ALL is that a large number of blast cells gather in the bone marrow and destroy the normal hematopoietic function of the bone marrow, resulting in the symptoms of anemia, infection and hemorrhage, and extramedullary infiltration can occur in the late stage. ALL accounts for about 80% of childhood leukemia, with the highest incidence in children aged 1 to 4 years. TCM network pharmacology, as a new mode of modern TCM research and development, contains the holistic concept of TCM, including TCM unique diagnosis and treatment ideas and rich clinical experience, and constitutes a research mode characterized by “network and “system.” A large number of clinical studies have shown that MSMY has a significant effect on ALL. However, the mechanism of action of MSMY in the treatment of ALL is still unclear. Therefore, in this study, network pharmacology was used to explore the possible mechanism of MSMY in the treatment of ALL, and to provide new theoretical support for the clinical treatment of ALL.Modified shengmaiyin (MSMY) is composed of Shengmai Powder, Radix Glycyrrhizae, Radix Astragali, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herba Solani Nigri and Radix Ampelopsis. Shengmai Powder was first recorded in Yi Xue Qi yuan and recorded in Leukemia Treatment of TCM. It has the effect of replenishing qi and nourishing yin. Modern pharmacological studies have shown that Shengmai Powder can improve the immune function of ALL patients and promote bone marrow hematopoiesis. It also protects the heart, liver, brain and other important organs, and reduces the myocardial toxicity caused by chemotherapy drugs.https://old.tcmsp-e.com/tcmsp.php) as the screening criteria, combined with literature data mining and data integration, and the targets of related active components were predicted. Oral Bioavailability (OB) and Drug Likeness index were used as the relevant indicators for screening, and the chemical components with OB ≥ 30% and Drug Likeness ≥ 0.18 in the database were set as candidate active ingredients and their target proteins were obtained. TCMSP and Swiss target prediction database (http://www.swisstargetprediction.ch/) were used for active ingredient target prediction. At the same time, the UniProt database (https://www.uniprot.org/) established for human species was used to query the screening targets, and the genes of these targets were normalized.The active components of MSMYs were retrieved by using TCM Systematic Pharmacology Database and Analysis Platform  and DisGeNET (https://www.disgenet.org/), using the keyword “acute lymphoblastic leukemia,” GeneCards were screened for Relevance score ≥ 20. To remove the duplicate gene targets, UniProt database was used to query the screening targets, and the above targets were normalized.ALL-related targets were obtained by searching GeneCards (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to screen the targets of “acute lymphoblastic leukemia” and “MSMY,” and the intersection targets of ALL and MSMY were obtained. At the same time, the intersection target points are visualized and analyzed.The Venny 2.1 online mapping platform (https://cn.string-db.org/), the species was defined as Homo sapiens, the protein interaction was obtained, the interaction threshold was set as “highest confidence” (>0.9), and the free protein in the results was hidden to obtain the PPI relationship between MSMY and ALL. Then the PPI network diagram was drawn by Cytoscape v3.9.1 software to screen the core targets, and the data of the core targets were visualized to construct the PPI network diagram.PPI network can reveal the topological structure characteristics and functional organization mechanism of protein complexes and functional modules in biological processes (BP) and clarify the molecular correlation between diseases and drugs. Import the obtained intersection targets into the STRING11.5 database (https://david.ncifcrf.gov/) was used for GO functional annotation and KEGG pathway enrichment analysis. Select “Homo species” on the DAVID platform; To further analyze the BP, cellular component, molecular function and signaling pathway of MSMY related to ALL treatment. The results were visualized and analyzed by bioinformatics online platform (http://bioinfo.seu.edu.cn/xxpt/).For the core targets of the screening, the DAVID data platform , and other settings were default settings. The PPI network relationship was obtained by hiding the free protein in the results, as shown in Figure , and othThe GO functional enrichment analysis of 59 intersected core targets was performed on the DAVID online platform, and 567 GO enrichment items were obtained, including 454BP, 36CC, and 77MF. According to the number of enriched genes, the top 10 items were selected for visual analysis, as shown in Figure The DAVID online platform was used to analyze the KEGG pathway enrichment of core intersection targets, and genes were enriched in 143 signaling pathways. According to the gene enrichment ranking, 30 signaling pathways were selected for visual analysis, as shown in Figure  ALL generally responds well to chemotherapy, and current dose-intensification strategies for ALL can significantly improve patient mortality, with 5-year overall survival currently estimated at 85% in developed countries and 35% in developing countries. However, early mortality, high side effects of chemotherapy and high recurrence rate are still the main problems.ALL is the most common type of leukemia with abnormal proliferation of hematopoietic stem cells. It is also a tumor disease with high incidence in children and adolescents. At present, it is divided into 3 types according to the severity of the disease: low-risk, intermediate-risk and high-risk. The pathogenesis of ALL is complex, involving multiple factors. Professor Sun Weizheng, Department of Hematology, the First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, believes that ALL is due to congenital deficiency or acquired malnutrition, the 6 exogenous pathogens cause the deficiency of viscera, and the toxin enters by taking advantage of the deficiency, causing qi stagnation and blood stasis in the human body, phlegm and blood stasis form the disease, and internal deficiency is the root cause, so the treatment should be based on the rise and fall of pathogens and healthy qi to “strengthen the healthy qi” and “eliminate pathogens.” Through the above understanding, Sun Weizhengjiao created MSMY on the basis of Shengmai San through many years of clinical experience and clinical efficacy observation. The prescription is composed of ginseng, Schisandra chinensis, Ophiopogon japonicus, Astragalus membranaceus, licorice, Hedyotis diffusa, Scutellaria barbata, Solanum nigrum and Ampelopsis japonica. Its efficacy is to benefit qi and promote body fluid, clear heat and detoxify. Radix Astragali, Radix Ginseng, Fructus Schisandrae Chinensis, Radix Ophiopogonis, etc. Are compatible to regulate and invigorate qi, blood, yin and Yang; Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herba Solani Nigri, Radix Ampelopsis, etc. Clear away heat and toxic materials; Radix Glycyrrhizae tonifies deficiency, regulates the middle warmer, and harmonizes all medicines in the whole prescription. Modern pharmacological studies have found that Shengmaisan can improve the immune function of ALL patients and promote bone marrow hematopoiesis. At the same time, studies have found that Astragalus polysaccharide plays an important role in promoting hematopoiesis, enhancing immunity and regulating inflammation. Herba Hedyotidis Diffusae and Herba Scutellariae Barbatae are widely used in various tumors. The main anti-tumor active components (flavonoids and anthraquinones) of Herba Hedyotidis Diffusae can induce apoptosis of leukemia cells, increase the specific immune function and nonspecific immune function of the body, and have a certain attenuation effect on tumor chemotherapy drugs. Diterpenoids, flavonoids and polysaccharides in Scutellaria barbata have the effect of treating leukemia by inhibiting the proliferation of tumor cells, inhibiting the formation and metastasis of tumor cells, regulating immunity, and inducing the apoptosis of tumor cells.TCM has outstanding performance in the synergistic treatment of ALL, which can improve the side effects of chemotherapy and improve the quality of life of patients. Moreover, ALL is classified into the category of “consumptive disease” and “blood syndrome” according to the clinical manifestations of TCM, with the accumulation of pathogenic toxin as the main feature, which can be divided into 4 types: deficiency of both qi and yin, excessive heat toxin, phlegm nucleus and accumulation of symptoms. Among them, about 90% of ALL is the accumulation of pathogenic toxin and deficiency of both qi and yin.Twelve main active components of MSMY in the treatment of ALL were screened out by TCM compound network pharmacology research method, comprising quercetin, Ophiopogon dihydroisoflavone E, homophylline, kaempferol, baicalein, muping aristolochic acid amide, N-coumaroyl tyramine, licochalcone A, red door blue phenol, Ophiopogon dihydroisoflavone B, beta-carotene and naringenin.,12 A number of studies have found that dihydroisoflavone E of Ophiopogon japonicus shows a variety of pharmacological activities, such as cardiovascular protection, anti-inflammatory, anti-cancer, antioxidant, immunomodulation, etc.; Homoplanin can play an anti-tumor role by inhibiting tumor cell proliferation, inducing tumor cell apoptosis, invasion and metastasis, tumor angiogenesis and other mechanisms, has multiple targets and a wide anti-tumor spectrum, plays a role in a variety of tumors, has weak toxicity to normal cells. At the same time, the combination of homo-plantain and a variety of anti-tumor drugs can significantly enhance the cytotoxicity of anti-tumor drugs to tumor cells. Kaempferol is the most effective scavenger of active oxygen free radicals, but its cytostatic effect and apoptosis-mediated effect are not as good as those of kaempferol.Among them, quercetin, Ophiopogon japonicus dihydroisoflavone E, homoplantain, kaempferol and baicalein are all flavonoids. Flavonoids have the effects of inhibiting proliferation, inducing apoptosis, blocking cycle, inhibiting angiogenesis, increasing the production of reactive oxygen species and inhibiting tubulin function in leukemia cells. Modern pharmacological studies have shown that quercetin can inhibit platelet aggregation, significantly inhibit platelet aggregation induced by thrombin and platelet activating factor, significantly inhibit the effect of cancer promoters, inhibit the growth of malignant cells in vitro, resist tumors, inhibit the expression of inflammatory mediators, and mediate cell signal transduction; Quercetin can inhibit the malignant proliferation of leukocytes, restore the hemogram to a relatively normal level, and has a significant inhibitory effect on ALL. Glucocorticoid resistance is a major driver of treatment failure in ALL, and AKT1 kinase acts as a major negative regulator of glucocorticoid receptor protein activity.AKT1 impairs glucocorticoid-induced gene expression by directly phosphorylating the glucocorticoid receptor at the S134 position and blocking glucocorticosteroid-induced translocation of the receptor to the nucleus.Vascular endothelial growth factor A regulates the transendothelial migration of microvascular endothelial cells, VEGF mediates leukemic cell entry into the central nervous system and leptomeningeal infiltration, and VEGF plays a role by activating the VGEFR family to regulate vascular growth and protect leukemic cells from apoptosis.Apoptosis is a kind of normal death under the control of relevant genes in order to maintain the homeostasis of cells. The proteins involved in the process of apoptosis include caspases and Bcl-2 (IAPs). The mode of action of CASP3 is that when cells are damaged, it mainly shows mitochondrial membrane damage, and cytochrome in mitochondria is released to the outside of cells, which activates CASP3 and induces apoptosis. CASP3 has attracted more and more attention as a target for the treatment of some cancers, and the study of CASP3 inhibitors is one of the effective methods for the development of anticancer drugs.In this study, 27 core targets were predicted for MSMY in the treatment of ALL, including triptolide, RAC-alpha serine/threonine-protein kinase (AKT1), vascular endothelial growth factor A, Caspase-3 (CASP3), Myc proto-oncogene protein and estrogen receptor 1. Among them, triptolide is a powerful tumor suppressor and one of the popular targets for the treatment of cancer. Activation of p53 can play a certain role in anti-cancer. P53 gene can play a role in inducing apoptosis of leukemia cells by inhibiting the activity of telomerase reverse transcriptase and telomerase and inhibiting the expression of Bcl-2 gene. MAPK signaling pathway is a process that transduces extracellular stimuli into cells and their nuclei and causes cellular biological responses. Ras/Erk signaling pathway is the most widely studied MAPK signaling pathway. Ras activates MAPK cascade and participates in the regulation of cell proliferation and differentiation. MAPK signal transduction pathway plays an important role in the pathogenesis of leukemia by mediating apoptosis inhibition signals to cause malignant transformation of cells, excessive proliferation and apoptosis inhibition. IL-7 has the ability to inhibit spontaneous apoptosis and promote the proliferation of ALL cells in vitro, which may be achieved by activating PI3K/Akt signaling pathway, up-regulating Bcl-2 and down-regulating P27kip1. Therefore, the treatment of ALL with MSMY may be highly related to signaling pathways, and relevant molecular experiments should be strengthened to explore the mechanism of action.GO and KEGG enrichment analysis showed that MSMY treatment of ALL may be related to the regulation of apoptosis, positive regulation of gene expression and protein binding. KEGG enrichment analysis involved in cancer, phosphatidylinositol 3 kinase-protein kinase B, MAPK and IL-17 signaling pathways. PI3K-Akt plays a key role in the prevention and treatment of ALL. Abnormal activation of PI3K-Akt is one of the most common pathogeneses of ALL. Down-regulation of PI3K-AKT can promote autophagy and apoptosis of ALL cells.In this study, on the premise that the clinical efficacy of MSMY in the treatment of ALL is confirmed, it lays the foundation for the following research, and uses the protein interaction network to explore the internal molecular mechanism of MSMY in the treatment of ALL. The results showed that the intrinsic mechanism of MSMY intervention in ALL may be related to the activation of PI3K-Akt, MAPK, IL-17 and other related signaling pathways mediated by MSMY core activity, which affects cell apoptosis and gene expression, so as to intervene in ALL.The chemical composition of TCM compound is complex, showing the characteristics of multi-component, multi-target and multi-way. With the development of systems biology, network pharmacology provides a feasible research method for exploring the internal principle of TCM active ingredients intervening diseases and the construction of TCM multi-target precise treatment mode, which plays a promoting role in the development of TCM.Conceptualization: Dongyu Guo, Jing Hao.Data curation: Dongyu Guo, Jing Hao.Formal analysis: Dongyu Guo, Mengyu Gu.Investigation: Yuting Zhao.Methodology: Dongyu Guo, Mengyu Gu, Fuquan Du.Project administration: Dongyu Guo, Fuquan Du.Resources: Dongyu Guo, Yuting Zhao, Mingjie Gao.Software: Fuquan Du, Yuting Zhao.Supervision: Dongyu Guo, Mingjie Gao.Validation: Dongyu Guo, Yuting Zhao, Mingjie Gao.Visualization: Dongyu Guo, Mingjie Gao.Writing – original draft: Dongyu Guo, Jing Hao.Writing – review & editing: Dongyu Guo.


To explore the molecular mechanism of Simiao Decoction (SMD) intervening atherosclerosis (AS). The main components and potential mechanisms of SMD remain unknown. This study aims to initially clarify the potential mechanism of SMD in the treatment of AS based on network pharmacology and molecular docking techniques. The principal components and corresponding protein targets of SMD were searched on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and the compound-target network was constructed by Cytoscape3.9.1. AS targets were searched on DrugBank, OMIM, and TTD databases. The intersection of compound target and disease target was obtained and the coincidence target was imported into STRING database to construct a protein–protein interaction network. We further performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis on the targets. The molecular docking method was used to verify the interaction between core components of SMD and targets. We created the active compounds-targets network and the active compounds-AS-targets network based on the network database containing Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, DrugBank, OMIM, and TTD. We discovered that the therapy of AS with SMD involves 3 key substances—quercetin, kaempferol, and luteolin—as well as 5 crucial targets—ALB, AKT1, TNF, IL6, and TP53. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the shared targets involved a number of signaling pathways, including the advanced glycosylation end product-receptor of AGE signaling pathway in diabetic complications, Hepatitis B, Lipid and atherosclerosis, Chemical Carcinogenesis-Receptor Activation, and Pathways in Cancer. The molecular docking demonstrated that the binding energies of quercetin, kaempferol, and luteolin with 5 important targets were favorable. This study reveals the active ingredients and potential molecular mechanism of SMD in the treatment of AS, and provides a reference for subsequent basic research. It is characterized by localized inflammation, lipid accumulation, smooth muscle cell proliferation, apoptosis, necrosis, and fibrosis. Moreover, AS provides the pathophysiological underpinning for certain cardiovascular illnesses. One of the most common diseases. The pathological process of AS mainly involves lipid deposition, endothelial damage, foam cell accumulation, and rupture of unstable plaques, which are prone to rupture and promote thrombosis, such as coronary artery disease and ischemic stroke. It’s the potential mechanism of cardiovascular adverse events. A number of pathophysiological processes in the body, including the inflammatory response, are linked to the development and progression of AS. Tumor necrosis factor α (TNF-α), proinflammatory factor interleukin (IL)-6, and AS can all be accelerated by inflammatory cytokines. Important clinical indications that represent the intensity of the inflammatory response in vivo are the levels of IL-1 and the anti-inflammatory cytokine IL-10. It is significant that traditional Chinese medicine has had outstanding outcomes in treating patients with various stages of AS while also significantly lowering the incidence of side effects and problems.,7Large and medium-sized arteries are the primary targets of the chronic illness atherosclerosis (AS). What is noteworthy is that network pharmacology is a new field that analyzes biological networks using the systems biology theory, chooses certain signal nodes, and develops multi-target therapeutic molecules. The Simiao Decotion (SMD)-active ingredients-AS target network and SMD-AS common target protein–protein interaction (PPI) network were created using the network pharmacology method. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the biological process, cell components, molecular functions, and key pathways, among other things, to systematically elucidate the mechanism of SMD in the treatment of AS.The practice of traditional Chinese medicine, which continues to be important in preserving peoples’ health, is getting more and more recognition on a global scale. In order to treat complicated diseases like cardiovascular disease, cancer and diabetes, modern medicine has adopted the notion of traditional Chinese medicine and uses compound medications.https://tcmspw.com/tcmsp.php). Based on parameters of pharmacokinetics, oral bioavailability ≥ 30% and drug-likeness ≥ 0.18 were selected as the conditions for screening active compounds to determine the active compounds in SMD.,12 All targets were introduced by the Uniprot database (https://www.uniprot.org/), and then a network of “Active compound targets” was built with Cytoscape 3.9.1 software.The compounds were searched by inputting “Huangqi,” “Danggui,” “Jinyinhua,” and “Gancao” as keywords on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , OMIM (https://omim.org/), and TTD (http://db.idrblab.net/ttd/). The Venn diagrams of the targets of SMD and AS were drawn out using R, and then the screening of common targets as important potential targets intervening AS in SMD was performed. The network of “active compound-AS-targets” was built using Cytoscape 3.9.1 software.AS related targets were searched by entering “Atherosclerosis” as keyword in DrugBank (https://string-db.org) platform with the mutual score set to “medium confidence 0.4” and the species set to “Human sapiens.” The topological parameters of the PPI network were acquired by the “Network Analysis” function in the Cytoscape 3.9.1 software, among which the greater degree value is the most important in the PPI network. The top 5 core targets were selected based on degree scores as important targets for SMD to interfere with AS. Then, the PPI networks of the 5 core targets were constructed by the STRING.11.5 platform and setting “Highest confidence 0.9” and the species “Homo sapiens.”In order to build a PPI network, we entered the targets intervening AS in SMD into the STRING.11.5 .,18 The functions of the GO and KEGG pathways were subjected to visualization analysis using ggplot in the R package.The GO functionhttps://www.rcsb.org/) and the PubChem database.,22 Chem Office software was used to perform mol2 format conversion and energy minimization in active compounds. After water molecules, original ligands, and polarized hydrogen from target proteins were removed via PyMOL software, the affinity and binding energy between the target protein and the active compounds were analyzed by Autodock Vina and Python scripts. Meanwhile, the binding energy (affinity) ≤ − 5.0 kJ/mol was a screening condition in this study.The top 5 targets of the degree value in the PPI network were selected as protein receptors, and the active compounds of the top 3 degrees were selected as ligands of the active compounds in the SMD intervening in AS for verifying the molecular docking, respectively. The target protein files in PDB format and the active compound files in SDF were obtained from the RSCB PDB database , Radix Angelicae sinensis (Danggui-DG), Lonicerae Japonicae Flos (Jinyinhua-JYH), and Licorice (Gancao-GC).Related targets of intervening Cytoscape 3.9.1 software was used to construct and analyze the active compound-target connection in SMD, and the resulting “active compounds-257 targets” network diagram is depicted in Figure AS in SMD as well as the corresponding active compounds were inputted into Cytoscape 3.9.1 software to build “active compounds-AS-targets” network diagram. After analysis of the mapping using the Network Analysis plug-in, it was found that the top 3 core active compounds with the degree value from high to low were MOL000098-quercetin, MOL000422-kaempferol, and MOL000006-luteolin. The topological parameters of the active core compounds mentioned above in the network of “active compounds-AS targets” are shown in Figure As seen in Figure P < .05), including 751 biological processes (BP), 88 cell compositions (CC), and 152 molecular functions (MF). In the study, the top 10 of BP, MF, and CC results were screened as per P-value for mapping a histogram, as shown in Figure The results of the analysis of the GO function showed that 991 enrichment results were obtained after screening (P < .05) were involved in Pathways in cancer, Lipid and AS, Chemical carcinogenesis - receptor activation, advanced glycosylation end product-receptor of AGE (AGE-RAGE) signaling pathway in diabetic complications, and Hepatitis B, etc. In the study, the top 10 signal pathways were selected according to the P value to map a path bubble diagram, as shown in Figure KEGG analysis showed that 169 signaling pathways (The protein receptors and active compounds corresponding to ALB (PID:1N5U), AKT1 PID: 1H10), TNF PID:7KP9), IL6 (PID: 1ALU), and TP53 PID: 4CZ7) were selected from the RSCB PDB database and PubChem database for molecular docking. The docking results of the 3 active core compounds and the 5 core protein receptors were all less than −10.0 kJ/mol, suggesting that the active core compounds in the SMD and core targets in AS had good binding capacity, respectively shown in Table CZ7 were , IL6 -induced foam cells and slow down the aging process by causing RAW264.7 cells to engage in STE20 like kinase (MST1)-mediated autophagy. AKT may control numerous downstream processes simultaneously, and after PI3K activation, AKT kinase activity is increased in a variety of growth factor receptor-mediated signal cascades. Cell growth and proliferation are mostly regulated by the PI3K-AKT signal pathway, which frequently collaborates with other signal pathways in this regard. It has been demonstrated that TNF-related receptor inhibitors enhance vascular endothelial function and decrease the concentration of serum adhesion molecules IL-6 is an inflammatory cytokine that can induce the death and necrosis of smooth muscle cells and vascular endothelial cells, which can result in the development and rupture of atherosclerotic plaque and even thrombosis. By inhibiting TP53 expression, miR-652-3p regulates the lipid metabolism of macrophages to reduce AS.We gathered disease targets for AS in the DrugBank, OMIM, and TTD databases. We detected 217 shared targets between AS and SMD. These targets are thought to be possible AS and SMD therapy targets. We built a PPI network expressing protein-protein interaction to investigate the key target. According to the PPI network data, the main targets may include ALB, AKT1, TNF, IL6, and TP53. ALB performs physiological tasks include transporting and binding both endogenous and foreign materials, preserving the osmotic pressure of plasma colloid, neutralizing free radicals, and influencing the permeability of arterial blood vessels. Advanced glycosylated ALB can cause endoplasmic reticulum dysfunction, which can increase the risk of AS. KEGG enrichment analysis results show that Pathways in cancer, Lipid and AS, Chemical carcinogenesis - receptor activation, AGE-RAGE signaling pathway in diabetic complications, and Hepatitis B, etc. AS and tumorigenesis may be linked by lectin-like oxidized ox-LDL receptor-1, indicating that inhibiting lectin-like oxidized ox-LDL receptor-1 may be a promising approach to treating both AS and malignancies. Via the suppression of oxidative stress, a combination of vitamin D receptor and retinoid X receptor agonists successfully treated diabetic AS, and it is possible that vitamin D receptor activation is partially responsible for the preventative actions of retinoid X receptor agonist. Peroxisome proliferator-activated receptor α, a nuclear receptor, can lessen AS and inhibit the development of macrophage foam cells, vascular inflammation, vascular smooth muscle cell migration and proliferation, plaque instability, and thrombosis. By activating Nox-1, nuclear factor κB, and other signaling pathways, the AGE-RAGE signaling pathway in diabetes complications can increase the production of reactive oxygen species, promote oxidative stress, and induce the occurrence and development of AS. Conversely, reducing the production of reactive oxygen species can decrease vascular calcification. Untreated HBeAg-negative chronic Hepatitis B is a unique risk factor for subclinical AS and carotid plaques.We used GO analysis and KEGG enrichment analysis to investigate the mechanism of SMD in the treatment of AS. The GO findings revealed that the target genes were mostly rich in biological activities, BP mainly covered positive regulation of transcription from RNA polymerase II promoter, positive regulation of gene expression, and response to drugs, estradiol, exogenous stimulation, etc. CC generally were related to extracellular space, cytosol, and nucleoplasm. MF mainly included protein binding and enzyme binding. Not only is catalytically active gamma-glutamyl transferase present in atherosclerotic plaques, but there is also a correlation between gamma-glutamyl transferase activity and markers of plaque instability, suggesting that the enzyme plays a direct role in the pathophysiology of AS and associated clinical events by encouraging pro-oxidant reactions.Based on the above results of network pharmacological research, specifically the screening results of compound active ingredients and the screening results of targets in PPI network, we screened 3 important bioactive components and 5 representative targets in order to investigate the potential molecular mechanism of SMD in treating AS. ALB, AKT1, TNF, IL6, and TP5 protein molecules are docked with quercetin, kaempferol, and luteolin, respectively, to confirm our network pharmacological prediction results. The docking findings revealed that all 3 substances could attach to protein with good efficiency, with quercetin and TNF having the lowest binding energy and the most stable combination. It can be used as a further research direction.There are still some limitations to this study. In this study, we investigate the targets of SMD in treatment of AS using network pharmacology and molecular docking techniques, the only modern bioinformatics methods. Quercetin, kaempferol, and luteolin have been identified as the 3 most significant bioactive components of SMD in treating AS, although they are unable to fully represent SMD. ALB, AKT1, TNF, IL6, and TP53 are also the primary targets.Some of the results of our research are less in previous studies. However, the accuracy and timeliness of database data need to be scientifically confirmed, and the present network information technology has to be further enhanced. We might not include in our study any chemicals or targets that have not been verified and recorded. In addition, we must continue to think about pharmacodynamic studies. We think there is a lot of space for development and study because the effects and mechanisms of these possible active components on AS haven’t been clarified and proven.Based on network pharmacology and molecular docking technology, this study conducted a preliminary exploration of the main chemical components and potential mechanism of SMD in the treatment of AS. Our research shows that quercetin, kaempferol, and luteolin may be the main active ingredients of SMD, and the protein target ALB, AKT1, TNF, IL6, and TP53 may be a potential therapeutic target of SMD in the treatment of AS. And it may play a therapeutic role through Pathways in cancer, Lipid and AS, Chemical carcinogenesis - receptor activation, AGE-RAGE signaling pathway in diabetic complications, and Hepatitis B, etc. In addition, our study will provide a reference for further research on the mechanism of SMD in the treatment of AS.We thank Yihui Chai for article revision/review; Wen Li and Liancheng Guan for assistance in data curation; and Yizi Fan for help with visualization.Data curation: Qian Li, Wen Li, Liancheng Guan.Funding acquisition: Yunzhi Chen.Project administration: Yunzhi Chen.Visualization: Yizi Fan.Writing – original draft: Qian Li.Writing – review & editing: Yihui Chai.


Traditional Chinese medicine suggests that Ginseng and Astragalus Decoction (GAD) may effectively treat postmenopausal osteoporosis (PMO). However, the exact mechanism of action for GAD remains unclear. This study aims to utilize network pharmacology and molecular docking technology to explore the potential mechanism of GAD in treating PMO. The main chemical components of GAD were identified by consulting literature and traditional Chinese medicine systems pharmacology database. GeneCards and online mendelian inheritance in man were used to identify PMO disease targets, and Cytoscape 3.8.2 software was used to construct a herb-disease-gene-target network. The intersection of drug targets and disease targets was introduced into the search tool for the retrieval of interacting genes platform to construct a protein-protein interaction network. Additionally, we further conducted gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses, followed by molecular docking between active ingredients and core protein targets. We have identified 59 potential targets related to the treatment of PMO by GAD, along with 33 effective components. Quercetin and kaempferol are the compounds with higher degree. In the protein-protein interaction network, IL6, AKT1, and IL1B are proteins with high degree. The enrichment analysis of gene ontology and KEEG revealed that biological processes involved in treating PMO with GAD mainly include response to hormones, positive regulation of phosphorylation, and regulation of protein homodimerization activity. The signal pathways primarily include Pathways in cancer, PI3K-Akt signaling pathway, and AGE-RAGE signaling pathway. Molecular docking results indicate that kaempferol and quercetin have a high affinity for IL6, AKT1, and IL1B. Our research predicts that IL6, AKT1, and IL1B are highly likely to be potential targets for treating PMO with GAD. PI3K/AKT pathway and AGE-ARGE pathway may play an important role in PMO. Due to decreased estrogen secretion, postmenopausal women experience weakened inhibition of osteoclasts due to changes in immune status and the release of reactive oxygen species.,5 This ultimately increases the risk of fracture, unnecessary bone pain, and other symptoms. Current treatment methods for PMO include raising estrogen levels, supplementing calcium, promoting calcium absorption, stimulating osteogenesis and inhibiting osteoclasts; however, these drugs have limited efficacy in improving bone metabolism and may cause adverse reactions. Therefore, there is an urgent need to explore safer and more effective treatment strategies for PMO. Natural products are gaining attention due to their relative safety and potential anti-osteoporotic effects.,14 Traditional Chinese medicine classifies PMO as “bone withered” and believes that “kidney and vital essence deficiency” are the main pathogeneses of PMO. Kidneys govern bones by generating marrow; kidney deficiency leads to passive vital essence depletion which results in increased bone fragility leading ultimately to PMO.Postmenopausal osteoporosis is a common metabolic bone disease in women. Although traditional Chinese medicine has long used ginseng for postmenopausal osteoporosis treatment, it lacks clinical analysis and experimental demonstration. Astragalus (AS) is a leguminous plant known for its direct role in tonifying the kidney according to ancient Chinese medicine books. Rihuazi Materia Medica records AS as being able to “invigorate qi and strengthen muscles and bones.” Clinical studies have demonstrated that AS can increase lumbar bone density, inhibit bone absorption, maintain positive balance by adjusting bone metabolism, and prevent bone loss.,17 Traditional Chinese medicine has a history of using AS for treating osteoporosis; however, research on its mechanism remains deficient.Ginseng is a highly valued medicinal material in traditional Chinese medicine due to its ability to tonify the kidney and replenish vital essence. Studies have shown that ginseng extract can improve symptoms of osteoporosis and arthritis in women. The goal of network pharmacology research is to systematically address scientific challenges at multiple levels. Interestingly, this theory aligns with the concept of preventive treatment in traditional Chinese medicine known as syndrome differentiation. By analyzing existing databases, network pharmacology can study traditional Chinese medicines and diseases. It offers a novel approach to overcome limitations in basic research on traditional Chinese medicine.Traditional Chinese Medicine has long used GAD to treat PMO. The combination of Ginseng and AS usually has a synergistic effect. However, this method only exists in ancient Chinese medical books and passed down experiences, lacking mechanism research. Due to the complexity of traditional Chinese medicine components, multiple targets, and broad signaling pathways, in-depth research is challenging. Network pharmacology is an emerging analytical strategy that uses virtual computing and database retrieval. It employs techniques like high-throughput screening, network visualization, and analysis to uncover complex biological network relationships involving drugs, genes, targets, and diseases. This helps predict drug mechanisms and provides references for understanding drug effects.Based on this situation, we established an herb-disease-gene-target network. Then we obtained the main signaling pathways and biological functions through gene ontology (GO) analysis and Kyoto encyclopedia of gene and genome (KEGG) enrichment analysis. Finally, molecular docking technology was used to verify the proposed functional components and major targets. This study screened active ingredients with practical value in GAD through network pharmacology, determined their target roles and interactions while predicting the therapeutic mechanism of GAD against PMO. This provides useful reference for future research in this field.Ethical approval was waived or not necessary, all procedures performed in studies do not involve human participants or animals.http://tcmspw.com/tcmsp.php, accessed in March, 2023) to identify the active components of GAD. Our selection criteria were based on absorption, distribution, metabolism and excretion processes. Specifically, we screened for ingredients with oral bioavailability ≥30% and drug-like properties ≥0.18 in order to obtain appropriate active compounds.,22We utilized the traditional Chinese medicine systems pharmacology database (TCMSP) platform  to standardize target and gene symbols. To obtain disease targets, we entered the keyword “postmenopausal osteoporosis” into both online mendelian inheritance in man  and Genecards  databases.,24 We then merged the targets from both databases, removed duplicates, and collected the remaining targets as PMO targets. Using Cytoscape 3.8.2 network visualization software, we constructed a network of effective components and action targets for “herb - disease - gene - target.” The network analyzer plug-in was used to analyze network characteristics and clarify interactions between effective components and targets.After identifying the effective target of GAD in the TCMSP database, we utilized the Uniprot database  was used to predict the interaction between proteins. The protein-protein interaction network (PPI network) was created by introducing overlapping targets. Cytoscape was used to construct the core PPI network.The search tool for the retrieval of interacting genes/proteins (STRING) . The GO analysis identified relevant biological processes, cellular components, and molecular functions (MF). Additionally, KEGG enrichment analysis enabled us to identify significant signaling pathways involved in these biological processes. The P value cutoff was set at P < .01, indicating that a lower P value suggests a higher probability of the current result being a genuine enrichment outcome rather than a random event. In this study, we used −log 10  as the unit, and as this value increased, the reliability of the enrichment results also increased.To elucidate the biological functions and signaling pathways associated with PMO, we conducted GO and KEGG enrichment analysis using the Metascape database  and TCMSP database, followed by dehydration and hydrogenation before importing them into Autodock Tools (ver.1.5.6). The small molecule compound was then docked with protein as receptor using molecular docking method, selecting the most suitable conformation. Finally, we generated a 3D binding mode diagram using Pymol (ver.1.8.x).Molecular docking is a theoretical simulation method used in drug design to predict the binding modes and affinities of molecules by studying their interactions with receptors and ligands.According to the screening criteria, oral bioavailability ≥30% and drug-likeness ≥0.18 were set in the TCMSP database for screening. The results showed that a total of 33 active components of GAD complex were obtained, and the details were shown in table We conducted a screening of PMO disease-related targets in online mendelian inheritance in man and GeneCards databases, following standard procedures and removing repeated targets. This yielded 1224 potential targets. We then sorted the predicted results from TCMSP and literature review, removed duplicates, matched them with corresponding disease targets, resulting in 59 core targets. A Venn diagram  analysis to determine the top 2 compounds, namely quercetin and kaempferol, for docking with the top 3 proteins IL6, AKT1, and IL1B. The best docking image of the receptor-ligand complex is presented in Figure The results indicate that quercetin and kaempferol can effectively interact with IL6, AKT1, and IL1B. Specifically, IL6 forms hydrogen bonds with quercetin through GLN-41, GLN-40, and LYS-170 Fig. A, while ,32 Our molecular docking results reveal that the compounds we screened have high affinity with the core target, as their binding energies are all <−5 kcal/mol. The specific docking energies can be found in Table The affinity between the receptor and ligand increases as the binding energy decreases, resulting in a more stable conformation. A binding energy of <−5 kcal/mol is generally considered indicative of good binding activity and affinity. Other studies have shown that quercetin alleviate ovariectomy-induced osteoporosis by modulating autophagy and apoptosis in rat bone cells. However, the current in vitro research mainly focuses on the prevention and potential mechanism of quercetin on PMO. For the first time, we demonstrated the role of quercetin in PMO through the mining of drug components, construction of network targets and molecular docking verification, which well corroborated previous views and provided a good reference for the next in vivo research or clinical trials of quercetin in the treatment of PMO. With regard to the effect of kaempferol on PMO, only studies have shown that kaempferol has an inhibitory effect on bone loss in rats with osteoporosis after ovariectomy, but there is still no study on its potential mechanism and clinical effect. Our research is the first to predict the mechanism of action of kaempferol in the treatment of PMO through network pharmacology and drug composition mining, which provides a strong reference basis for subsequent research, which is our significance.PMO is a metabolic disease that women are prone to suffer from after menopause. Once it is not treated in time, it may lead to fracture, which seriously threatens the life and health of postmenopausal women. The traditional Chinese medicine GAD has been used in the treatment of PMO for a long time, but its mechanism and target are still unclear. The main purpose of this research is to explore the potential mechanism of GAD in the treatment of PMO. Through the analysis of traditional Chinese medicine compound database and disease database and the construction of network diagram, we speculate that the main chemical components of GAD in the treatment of DF include quercetin and kaempferol etc. Animal research shows that quercetin rescued TNF-alpha-induced impairments in bone marrow-derived mesenchymal stem cell osteogenesis and improved osteoporosis in rats. IL1B is a member of the IL-1 family. It has been proved that IL1B can regulate the chronic inflammatory response of many diseases, such as Parkinson disease, amyotrophic lateral sclerosis idiopathic tremor Alzheimer disease. Research on the polymorphism of IL-1B has been carried out worldwide. At present, both in vitro and in vivo experiments also show that IL-1B can significantly affect cartilage destruction and bone resorption.–45 However, the research on the effect of IL1B on PMO is still rare, which may also be a future research direction.Through the construction of PPI network, we can find that IL6, AKT1, and IL1B are the core proteins of GAD in treating PMO. Some studies have shown that IL-6 is an important pathogenic factor of immune-mediated bone diseases such as rheumatoid arthritis and postmenopausal osteoporosis. IL-6 mediates the actions of osteoblasts and osteoclasts through sophisticated mechanisms, which reflect dual effects.–48 Recent studies have shown that PI3K/AKT pathway plays an important role in the process of cell apoptosis.,50 In addition, PI3K/AKT pathway is closely related to cell proliferation and apoptosis.,52 Furthermore, AKT and PI3K are generally downregulated when osteoporosis occurs, illustrating that the progression of osteoporosis might be associated with cell injury.,53,54 There are many other studies on the role of PI3K/AKT pathway in PMO, for example, silence of miR-483-5p can alleviate postmenopausal osteoporosis through PI3K/AKT pathway; Chlorogenic acid can prevent osteoporosis in ovariectomized rats through PI3K/Akt pathway. Therefore, PI3K/AKT pathway is an important pathway for the occurrence and development mechanism of PMO, and also an important medium for the treatment of PMO. Our research speculates that GAD treatment of PMO is probably achieved through PI3K/AKT pathway. This also confirms the prediction result of our PPI network, that is, AKT1 plays an important role in the treatment of PMO with GAD.KEGG analysis enrichment analysis results mainly include pathway in cancer, PI3K-Akt signaling pathway and AGE-RAGE signaling pathway. Previous studies have shown that quercetin, in addition to reducing osteoporosis by regulating autophagy and apoptosis of bone cells, also participates in many molecular mechanisms of cancer occurrence, which may be the main reason for the enrichment of pathways in cancer.–59 but the latest systematic review shows that AGE-RAGE signaling inhibitors are the latest therapeutic targets for age-related diseases such as osteoporosis, rheumatoid arthritis, etc. Another animal study showed that lycopene can regulate bone metabolism through AGE/RAGE pathway, thus delaying the progress of osteoporosis in mice. In addition, diabetes itself can damage the metabolism and function of bone cells, leading to an increased risk of brittle fracture, which may also be the embodiment of AGE-RAGE signaling pathway in PMO. Therefore, combined with our research, we believe that the research on AGE-RAGE signaling pathway should not be limited to the complications of diabetes, it may have effects on a variety of metabolic diseases, which may also be a future research direction.AGE-RAGE signaling pathway has been considered as an important signaling pathway for complications of diabetes,,63 we are not surprised by the results of GOBP. The research results of GOMF and GOCC also revealed some MFs of GAD in treating PMO, and also provided some reference for the pathogenesis of PMO.GOBP results suggest that the treatment of PMO by GAD is most likely related to the response to hormone. The analysis of GOMF and GOCC respectively suggested that the most important cell component was transcription regulator complex, and the most likely MF is the regulation of the activity of protein homodimerization. Since postmenopausal osteoporosis itself involves a series of hormone imbalance processes,Based on the above discussion, we can boldly guess that small molecules quercetin, kaempferol and protein IL6, AKT1, IL1B play an important role in the treatment of PMO by GAD. Therefore, we conducted molecular docking between the screening results of compound active ingredients and the main targets in PPI network to verify the predicted results of our network pharmacology. The docking results showed that the 3 compounds could bind well with protein, and their minimum binding energy was <−5 kJ/mol. Thankfully, the molecular docking results have well verified our prediction. It is precisely because the therapeutic effects and mechanisms of these potential active ingredients on PMO have not been explained and verified, so we believe that the development space and significance are great.This study still has some limitations. We used modern bioinformatics methods to explore the role of GAD in PMO through network pharmacology and molecular docking. However, at present, the network information technology needs to be further improved, and the accuracy and timeliness of database data need to be scientifically verified. Meanwhile, although quercetin and kaempferol are considered to be the most important bioactive components for treating GAD in PMO, they cannot fully represent the GAD. Therefore, pharmacodynamic and molecular biological experiments need to be considered to further verify our research results. Additionally, clinical studies are still required to determine the effectiveness of GAD in treating PMO. This is the focus of our next work.In this study, we first used the method of combining network pharmacology and molecular docking to clarify the mechanism of GAD in treating PMO. Quercetin and kaempferol may be the main effective compounds for treating PMO through GAD therapy. They may slow down the progress of osteoporosis by regulating cell autophagy, inhibiting bone loss, and inducing osteogenesis. In addition, we proposed that PI3K/AKT pathway and AGE/ARGE pathway may play an important role in postmenopausal osteoporosis and predicted the future research direction. In conclusion, this study provides theoretical support for further in-depth study of the mechanism of GAD in treating PMO, and provides possible future research directions and reference basis for the pathogenesis of PMO and new treatment methods. However, further clinical experiments and in-depth basic research are necessary to fully comprehend the therapeutic effects and molecular mechanisms of traditional Chinese medicine on PMO.The authors would like to thank all authors of references.Conceptualization: Wei Fan, Sheng-Rong Wan.Data curation: Wei Fan, Sheng-Rong Wan.Formal analysis: Wei Fan, Sheng-Rong Wan.Investigation: Zong-Zhe Jiang.Methodology: Zong-Zhe Jiang.Project administration: Zong-Zhe Jiang.Resources: Zong-Zhe Jiang.Software: Wei Fan, Zong-Zhe Jiang.Supervision: Sheng-Rong Wan.Validation: Sheng-Rong Wan.Visualization: Sheng-Rong Wan.Writing – original draft: Wei Fan, Zong-Zhe Jiang, Sheng-Rong Wan.Writing – review & editing: Wei Fan, Zong-Zhe Jiang, Sheng-Rong Wan.


To verify the clinical efficacy of Zhang’s Xibi formula (ZSXBF) and explain the mechanism underlying its therapeutic effect.Preliminary elucidation of the clinical efficacy of ZSXBF in treating KOA in self-control studies, exploration of its mechanism of action with network pharmacology methods, and validation in animal experiments.In clinical studies, ZSXBF administration effectively improved patient quality of life and reduce pain. Network pharmacology was used to explore the possible mechanisms underlying its treatment effect, and after verification in clinical experience and animal experiments, it was found that ZSXBF regulated the expression of immune-related proteins such as IL-17, ERK1, and TP53 in mouse knee joints.ZSXBF, which is a traditional Chinese medicine compound that is used to clear heat and detoxify, can effectively improve the clinical symptoms of KOA patients, and its underlying mechanism includes the regulation of human immune-related proteins.The online version contains supplementary material available at 10.1186/s13018-023-04453-6. Osteoarthritis (OA) is a major cause of pain and disability worldwide , 2. OA iAt present, there is no definite and effective drug for the treatment of OA, and its clinical treatment mainly involves symptomatic treatment, for example, the use of nonsteroidal anti-inflammatory drugs (NSAIDs) to relieve pain; however, when NSAID treatment is ineffective, there are almost no drugs available for follow-up treatment . The lacZhang’s Xibi formula (ZSXBF) is the result of many years of experience in clinical practice by a famous teacher of traditional Chinese medicine in Zhejiang Province. This compound prescription, which is mainly composed of traditional Chinese medicine that is used to clear away heat and toxic materials, is considered to exert good effects in the treatment of KOA. It is generally believed that the heat-clearing and detoxification functions of this medicine may be related to the inhibition of immune responses that are caused by inflammation in the human body –9.However, the components of ZSXBF and the mechanism underlying its treatment effects against KOA have not yet been elucidated. Therefore, this study aimed to predict the key active ingredients and potential pharmacological mechanisms of ZSXBF in the treatment of OA through clinical experience, network pharmacology, and animal experimental methods.This study was approved by the Ethics Committee of Hangzhou Fuyang Traditional Chinese Medicine Bone Injury Hospital (No. 2023-LW-LC-003).Data from 146 KOA patients who were treated with ZSXBF at Fuyang Bone Injury Hospital in Hangzhou City between January 1, 2021, and December 31, 2021, were collected. To evaluate clinical outcomes, visual analog scale (VAS) and Leqiesne’s score examinations were performed one week before (baseline) and one month after treatment.ZSXBF consists of 6 Chinese herbs, including Dang-gui (DG), Chuan-niuxi (CNX), Ze-lan (ZL), Huang-bo (HB), Tu-fuling (TFL) and Mao-rensheng (MRS). The detailed data of ZSXBF are shown in Table The chemical components of the traditional Chinese medicine herbs in ZSXBF were searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) databases. Oral bioavailability (OB) and drug likeness (DL) were set as the screening criteria. In addition, eligible putative targets of these potential active compounds were identified in the TCMSP databases.Using knee osteoarthritis as the key word, associated target genes were identified by a joint search of the Genecards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, Therapeutic Target Database (TTD), and DrugBank databases. All the collected target genes, which were in the form of abbreviations, were edited in the same Microsoft Excel table, and duplicates were removed.R was used to screen target genes that were shared by KOA and ZSXBF. Then, the STRING online tool was used to analyze the target genes to generate a protein‒protein interaction network (PPI). The enrich plot tool was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.https://pubchem.ncbi.nlm.nih.gov/) [http://www.rcsb.org/pdb/home/home.do). 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 grid box was centered to cover the domain of each protein and to accommodate free molecular movement. The grid box was set to 30 Å × 30 Å × 30 Å, and grid point distance was 0.05nm. Molecular docking studies were performed by Autodock Vina 1.2.2 (http://autodock.scripps.edu/).To analyze the binding affinities and modes of interaction between the drug candidate and their targets, AutodockVina 1.2.2, a silico protein–ligand docking software was employed . The molih.gov/) . The 3D All the botanical drugs in ZSXBF were provided by the First Affiliated Hospital of Zhejiang Chinese Medical University (Hangzhou China). After soaking in 10 volumes of distilled water for 1 h, DG, CNX, ZL, HB, TFL, and MRS were mixed  at a ratio of 6:15:15:9:25:30 for reflux extraction . Then, the solution was concentrated (3 g crude drug/mL).To determine the main chemical components of ZSXBF, high-performance liquid chromatography (HPLC) was used. We added 0, 1 ml, 2 ml, and 4 ml of ZSXBF soup to four round-bottomed flasks and added 20 ml of 70% methanol  solution. After weighing, the solution was ultrasonicated for 30 min, allowed to cool, and weighed again, and the extraction solvent was used to make up the lost weight. The mixture was shaken well and filtered, and the filtrate was taken to obtain the final product.An Ultimate ®UHPLC AQ-C18  chromatographic column was used. The mobile phase consisted of acetonitrile  and 0.2% acetic acid . The gradient elution procedure is shown in Table The botanical medicinal materials used in this study were sent to the Drug Testing Laboratory of Zhejiang University of Traditional Chinese Medicine Academy of Traditional Chinese Medicine for identification according to The Chinese Pharmacopoeia (2020 edition). The quality of ZSXBF was controlled, and the exact chemical composition was determined by ultra-performance liquid chromatography (UPLC). UPLC identified 18 chromatographic peaks representing 18 drug monomers  was performed on 10-week-old male C57BL/6 mice to establish the knee osteoarthritic model. Briefly, DMM surgery was performed on the right hind limbs of C57BL/6 mice. First, a 3 mm longitudinal incision was made on the medial part of the knee under anesthesia, and blunt dissection of the knee extensor muscles and patellar ligament (MMTL) was performed. Then, the MMTL was transected to destabilize the medial meniscus. Finally, the medial joint capsule was sutured, and the skin was closed. Sham surgery was also performed on C57BL/6 mice with a similar surgical approach but without manipulating the joint tissues.n = 6 in each group): the sham group, DMM group, and XBF group. In the XBF group, the drug was orally administered to the mice for 8 consecutive weeks  beginning on the day after DMM surgery.All the C57BL/6 mice were randomly divided into three groups . All the studies were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (20200622-07).3).All the mice were sacrificed at 8 weeks after surgery, and the right knee joints were collected. Then, microcomputed tomography (μ-CT) was used to analyze the knee joints. The area between the proximal tibia growth plate and the tibial plateau was chosen as the region of interest, and the parameters collected by μ-CT were percent bone volume , trabecular thickness , and bone mineral density  solution for 14 days, and embedded in paraffin. Then, 3 μm thick sections of the medial compartment of the joints were generated for Alcian Blue Haematoxylin/Orange G (ABH) staining to analyze gross cartilage structural changes. The thickness of the tibial cartilage was scored by three blinded observers.Immunohistochemistry (IHC) was performed to observe the expression of type II collagen , matrix metallopeptidase 13 , interleukin 17 , tumor protein p53 , mitogen-activated protein kinase 14 , and extracellular regulated protein kinase 1 .The baseline characteristics of the participants in the incidence and progression cohorts are shown in Table To evaluate the clinical efficacy of ZSXBF in the treatment of KOA, we scored the treatment outcomes of 146 patients. After treatment with ZSXBF, the VAS scores and Lequesne scores were significantly improved, indicating that after treatment with ZSXBF, pain levels improved, and walking and ability to perform activities of daily life improved (Table To prove the effect of ZSXBF treatment on cartilage and subchondral bone, which are the sites of injury in KOA, DMM surgery was performed on C57BL/6J mice, and ZSXBF was intragastrically administered for 8 weeks. After 8 weeks, knee joint tissue samples were taken for μ-CT scanning, and 3D reconstruction was performed to obtain representative images of the knee joints. The μ-CT scanning results showed that the DMM model group had significant subchondral bone sclerosis and cartilage loss and that the BMD, subchondral bone BV/TV, and Tb.Sp values were significantly different Fig. A, D, E. The Pharmacology Database and Analysis Platform database were used in this analysis. Six drugs in ZSXBF were identified in the TCMSP, and the TCMSP showed that CNX, DG, HB, TFL, ZL and MRS had 4, 2, 37, 15, 2 and 0 targets, respectively. After eliminating duplicate targets, 212 predicted target genes were identified. To determine the potential targets of OA, we searched 1817 OA-related target genes in the GeneCards, DrugBank, TTD, OMIM, and PharmGkb databases. To investigate the potential target genes of ZSXBF in the treatment of OA, Venn analysis was performed, and 114 genes were identified as overlapping genes between ZSXBF and OA targets (Fig. We used STRING to construct a PPI network to determine the key proteins in the treatment of OA with ZSXBF. The thresholds of betweenness, closeness, degree, eigenvector, information, LAC and subgraph were set in two steps based on the topology parameters calculated by Cytoscape. Finally, ten key proteins, including ESR1, AKT1, RELA, TP53, MAPK1, MAPK3, JUN, FOS, EGFR, and MAPK14, were identified (Fig. GO and KEGG pathway enrichment analyzes of 168 tandem targets were conducted with the database. Based on network pharmacological analysis, we found that the AGE-RAGE signaling pathway and PI3K-Akt signaling pathway may be the most relevant signaling pathways for the treatment of OA with ZSXBF Fig. A, B.Fig.To evaluate the affinity of the drugs in ZSXBF for their targets, we performed molecular docking analysis. The binding poses and interactions of the main drug candidate with the main protein were obtained with Autodock Vina v.1.2.2 (Additional file The network pharmacology results suggest the possible mechanism by which ZSXBF treats KOA. Combined with our understanding of ZSXBF TCM treatment, we aimed to verify whether ZSXBF regulates cellular immunity, and we performed IHC staining for IL-17A, TP53, MAPK14, and ERK1. The results showed that the expression levels of IL-17A, TP53, MAPK14 and ERK1 in the articular cartilage of DMM model mice were significantly higher than those in control mice, suggesting that the pathogenesis of knee osteoarthritis is closely related to the immune factor IL-17. The expression levels of IL-17A, TP53 and ERK1 in the treatment group were significantly lower than those in the DMM model group, and there was no significant difference in the expression level of MAPK14 (Fig. TCM is an ancient treatment method, and it has been widely accepted and applied in the treatment of knee osteoarthritis , 13. HowBefore verifying the mechanism, our study further elucidates the clinical efficacy of ZSXBF. Through a retrospective analysis of the clinical efficacy of ZSXBF, the agreement party, we found that ZSXBF can effectively relieve the pain of KOA patients and improve their quality of life according to Lequesne’s score. In this retrospective study, patients' own baseline levels were used as the control group.To compensate for the limitations of the self-control study, we further verified the therapeutic effect of ZSXBF in experimental animals in a random control study. The DMM model is a widely used and recognized mouse model of KOA , 15. We Then, the mechanism underlying the therapeutic effect of ZSXBF was analyzed through network pharmacology. In contrast to chemical drugs, TCM has complex components, and it has multiple effective compounds and targets. In recent years, a new method has been used to study the mechanism underlying drug treatment of complex diseases . FurtherThrough network pharmacology, we identified possible therapeutic targets of ZSXBF. These therapeutic targets were compiled and calculated from other researchers' studies on TCM components and KOA through a computer system. Some researchers studied CNX and found that it can protect against chondrocyte damage induced by IL-1β . ResearcChanges in the T-cell population have been observed in KOA, including an increase in the proportion of Th17 cells . A studyIn summary, in this article, we have preliminarily discussed the therapeutic effect and underlying mechanism of Zhang's prescription in the treatment of knee arthralgia. We believe that ZXBF has a clear therapeutic effect in clinical practice. After computer data analysis and experimental verification, we believe that the mechanism by which ZSXBF treats OA is related to IL-17, ERK1 and TP53. Combined with the published results of other researchers, we speculate that ZXBF can delay the progression of KOA by regulating the IL-17-mediated immune response.Additional file 1: Fig. S1. Binding mode of screened drugs to their targets by molecular docking. Binding mode of quercetin to TP53.


Introduction: Insulin has an effect on neurodegenerative diseases. However, the role and mechanism of insulin in vascular dementia (VD) and its underlying mechanism are unknown. In this study, we aimed to investigate the effects and mechanism of insulin on VD.Methods: Experimental rats were randomly assigned to control (CK), Sham, VD, and insulin (INS) + VD groups. Insulin was administered by intranasal spray. Cognitive function was evaluated using the Morris's water maze. Nissl's staining and immunohistochemical staining were used to assess morphological alterations. Apoptosis was evaluated using TUNEL-staining. Transcriptome and metabolome analyses were performed to identify differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs), respectively.Results: Insulin significantly improved cognitive and memory functions in VD model rats (p < 0.05). Compared with the VD group, the insulin + VD group exhibited significantly reduced the number of Nissl's bodies numbers, apoptosis level, GFAP-positive cell numbers, apoptosis rates, and p-tau and tau levels in the hippocampal CA1 region (p < 0.05). Transcriptomic analysis found 1,257 and 938 DEGs in the VD vs. CK and insulin + VD vs. VD comparisons, respectively. The DEGs were mainly enriched in calcium signaling, cAMP signaling, axon guidance, and glutamatergic synapse signaling pathways. In addition, metabolomic analysis identified 1 and 14 DEMs between groups in negative and positive modes, respectively. KEGG pathway analysis indicated that DEGs and DEMs were mostly enriched in metabolic pathway.Conclusion: Insulin could effectively improve cognitive function in VD model rats by downregulating tau and p-tau expression, inhibiting astrocyte inflammation and neuron apoptosis, and regulating genes involved in calcium signaling, cAMP signaling, axon guidance, and glutamatergic synapse pathways, as well as metabolites involved in metabolic pathway. Vascular dementia (VD) is a chronic progressive disease that is characterized by mental and cognitive impairment resulting from various cerebrovascular diseases including hypertension, diabetes, hyperlipidemia, cerebral infarction, cerebral hemorrhage, and chronic cerebral ischemia . Among tImpairment of brain energy metabolism is one of the main features of neurodegenerative diseases including VD, Parkinson’s disease (PD), and AD . PatientInsulin is an important hormone in regulating blood glucose levels. Recent studies have demonstrated that insulin and its receptors are present in the human and mammalian central nervous systems (CNS) . There iMulti-omics analysis, including genomics, transcriptomics, proteomics and metabolomics analysis, is an effective tool for finding new targets or mechanisms . Since tTo identify new theoretical VD-related targets of insulin treatment, the present study was designed to investigate the effect of insulin on bilateral common carotid artery ligation (2-VO) induced VD model rats and to combine and analyze the deregulated transcriptome and metabolome profiles of VD model rats treated with intranasal insulin.st postinjury day. The limbs of mice were fixed in the horizontal position and the neck was kept straight to ensure smooth breathing. A total of 1U/10 μL insulin was dripped alternately through both nostrils using a microdoser (10 μL). After the infusion, the mice remained in position for 5–10 s to ensure that the fluid was fully absorbed. The dose and duration of insulin treatment followed the studies conducted by (1) CK group: normal saline (0.9%), 0.5 mL/d;(2) Sham group: normal saline (0.9%), 0.5 mL/d;(3) VD group: normal saline (0.9%), 0.5 mL/d;(4) Insulin (INS) + VD group: 1 U/kg/d;All animal experiments were conducted at the Neuroscience and Behavioral Research Center of Changsha Medical College. The experimental procedure followed ethical standards and the Declaration of Helsinki, as well as national and international standards. The research procedure was approved by the Ethics Committee of Changsha Medical University (CSMUE-20220182). Sixty Sprague-Dawley (SD) rats  were purchased from Hunan Slyke Jingda Biotechnology Co., Ltd. The rats were housed under controlled conditions . After adaptive feeding for 7 days, rats were randomly assigned to 4 groups (n = 15): (1) control (CK) group, (2) sham group (3) VD group, (3) insulin (INS) + VD group. Rats in the CK group did not receive any treatment. Rats in the sham group underwent separation of the carotid artery without ligation. Rats in the other 2 groups underwent 2-VO. The 2-VO procedure was performed according to The Morris water maze test, which tests spatial navigation, was performed on the 50th −56th postinjury days. The procedure of the Morris water maze test followed the study conducted by Rats were peritoneally anesthetized with pentobarbital sodium (40 mg/kg). Six whole brain samples were placed in 4% paraformaldehyde for morphological analysis. The other 6 whole brains and 3 hippocampal tissues were frozen in liquid nitrogen and then placed in a −80°C refrigerator separately for further metabolomic and transcriptomic analysis.Nissl staining was used to determine the distribution of Nissl’s body in the CA1 region of the hippocampus. Tissue sections were placed in the following solutions in order: xylene (15 min), xylene (15 min), anhydrous ethanol (5 min), anhydrous ethanol (5 min), 90% alcohol, 75% alcohol (5 min), and tap water. Nissl’s stain (tolonium chloride) was added and incubated for 5 min. Tissue sections were then differentiated with 1% glacial acetic acid for 2–3 s. Finally, the sections were cleared with xylene for 5 min and examined under a microscope.3) were immersed in 20%–40% sucrose with 0.1 M phosphate buffer solution (PBS), precipitated sugar in a gradient at 4°C, and then stored in a −80°C refrigerator. 20 μm sections from each sample were collected and placed in culture plates filled with PBS , and then transferred to antifreeze cache solution for storage at −20°C. Warmed brain tissue sections were placed in a reaction tube containing 5% normal horse serum and incubated at room temperature for 2 h. Primary antibodies (Anti-p-tau (181) (1:500) ; anti-tau (1:500) ; anti-Aβ1-42 (1:800) ; anti-GFAP   were added and incubated at room temperature for 2 h and at 4°C overnight. Tissues were selected in biotinylated broad-spectrum IgG (PAN:5% horse serum = 1:200) and incubated at room temperature for 2 h. Biotin-vitalin-horseradish peroxidase (ABC) complex was added to 0.01 M PBST buffer and incubated at room temperature for 2 h. The tissues were placed in 0.05% diaminobenzidine (DAB) dye solution, and then 0.03% H2O2 was added sequentially after preincubation. The tissue patch was immersed in the hematoxylin dye solution for color observation. Sections were then dehydrated through a series of 80%, 90%, 100%, and 100% graded alcohols, cleared twice through xylene, and then embedded in neutral resin.Postfixed brain tissue blocks (0.5 cmhttps://imagej.en.softonic.com/). The integrated optical density (IOD) of immunoreactivity was analyzed using OptiQuant software .The tissue sections were sequentially washed in xylene Ⅰ (20 min), xylene Ⅱ (20 min), anhydrous ethanol Ⅰ (10 min), anhydrous ethanol Ⅱ (10 min), 95% alcohol (5 min), 90% alcohol (5 min), 80% alcohol (5 min), 70% alcohol (5 min), and distilled water. Then the cells were covered with protease K and incubated at 37°C for 15 min. The tissues were washed in PBS (pH = 7.4), covered with a film-breaking working solution, incubated at room temperature, and then washed in PBS (pH = 7.4). TUNEL staining was performed using a one-step TUNEL cell apoptosis detection kit . The cells were observed using a fluorescence microscope . Morphological changes in the hippocampal CA1 region were measured using Image-Pro Plus 5.1 software  . RNA purity and concentration were determined by ultraviolet spectrophotometry . RNA was selected with an optical density (OD) between 1.8 and 2.0.The mRNA was enriched with oligo (dT) using magnetic beads, and the mRNA was cleaved into short fragments by adding the cleavage reagent. The fragmented mRNA was used as a template to synthesize one cDNA strand with six-base random primers, and then a two-strand synthesis reaction system was prepared to synthesize double-stranded cDNA, and the double-stranded cDNA was purified using a kit . The purified double-stranded cDNA was then terminally repaired, a tail was added, and a sequencing linker was connected. Then, fragment size selection was performed, and PCR amplification was performed. After the constructed library was qualified by an Agilent 2,100 Bioanalyzer . An Illumina HiSeqTM 2,500 sequencer  was used for sequencing. Illumina sequencing was performed at CapitalBio Technology Co., Ltd. .https://daehwankimlab.github.io/hisat2/). Gene expression levels were calculated using the fragments per kilobase of transcript per million fragments mapped (FPKM) method. Differential expression analyses between the two comparisons  were performed using the limma package (1.10.1). Gene with a |log2 fold change (FC)|≥1 and an adjusted p-value < = 0.05 was considered differentially expressed gene (DEG). Cluster analysis of differential gene expression patterns was performed using the R package. Gene Ontology (GO) annotation  (http://www.ebi.ac.uk/QuickGO/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment (http://www.genome.jp/kegg/) analyses of DEGs were applied.Clean sequences were compared to reference genome sequences using HISAT2 software  on an ABI 7,500 system . β-Actin expression levels were used as standardized internal control. RNA expression levels were calculated using the 2−ΔΔCt method. Primer sequences are listed in Three hippocampal RNA samples from the CK, VD, and INS + VD groups were tested. RNA was reverse transcribed into cDNA using Prime-Script™ RT Master Mix . qPCR was performed using SYBRSix hippocampal samples from 3 groups were homogenized using a grinder (30 HZ). A 400 μL solution  containing an internal standard was added. A 300 μL supernatant was collected and stored at −20°C for 30 min. The samples were then centrifuged at 12,000 rpm for 3 min (4°C). A 200 μL aliquot of the supernatant was subjected to LC-MS analysis with the following analytical conditions: UPLC: column, Waters ACQUITY UPLC HSS T3 C18 ; column temperature, 40°C; flow rate, 0.4 mL/min; injection volume, 2 μL; solvent system, water (0.1% formic acid): acetonitrile (0.1% formic acid); gradient program, 95:5 V/V at 0 min, 10:90 V/V at 11.0 min, 10:90 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 14.0 min. The metabolome profiling analysis was performed at CapitalBio Technology Co., Ltd. .www.r-project.org), respectively. DEMs were selected by variable importance in projection (VIP) (VIP ≥ 1), p-value  and absolute Log2FC (|Log2FC| ≥ 1.0) in two-group analysis. The orthogonal partial least-squares-discriminant analysis (OPLS-DA) results were generated from the R package . Cluster analysis of differentially expressed metabolite (DEM) expression patterns was performed by using the R package. DEMs were annotated using the KEGG compound database (http://www.kegg.jp/kegg/compound/).Unsupervised principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed using PRCOMP and Complex Heatmap in the R package .F test was used to compare components. p < 0.05 indicated a statistically significant difference.SPSS 25.0 was used for statistical analysis . For continuous variables, the normal distribution test was performed first. Measurement data conforming to a normal distribution were expressed as the mean ± standard deviation (±s), and one-way ANOVA was used to compare groups. If the variances were homogeneous, the least Significant Difference (LSD) test was used to compare between groups. When variances were not homogeneous, the Welch approximate p > 0.05). The mean escape latency in the VD group significantly increased compared to the CK group (p < 0.05). After treated with insulin, the mean escape latency in the INS + VD group significantly decreased compared with the VD group (p < 0.05). The number of hidden platform crossings in the CK, Sham, VD, and INS + VD groups were 4.3 ± 0.52, 4.0 ± 0.31, 1.2 ± 0.36, and 3.2 ± 0.37, respectively. The number of crossing the hidden platform crossings in the Sham group not significantly changed compared with that of the CK group (p > 0.05). The number of hidden platform crossings in the VD group significantly reduced compared to the CK group (p < 0.05). After treated with insulin, the number of hidden platform crossings in the INS + VD group significantly increased compared with that of the VD group (p < 0.05). . . The resp > 0.05). The IOD of Nissl’s body in the VD group (15.12 ± 3.2) was significantly reduced compared to the CK group (25.18 ± 5.1) (p < 0.05). After treated with insulin, the IOD of Nissl’s body in the INS + VD group (22.59 ± 2.7) significantly increased compared to the VD group (15.12 ± 3.2) (p < 0.05) (As shown in  < 0.05) .p < 0.05), indicating that ischemia might induce neuron apoptosis. And the proportion of TUNEL-staining positive cells in the hippocampal CA1 region exhibited significantly reduced in the INS + VD group compared to the VD group (p < 0.05), indicating that insulin might inhibit apoptosis . Compared to the CK group, GFAP IOD were significantly increased in VD group  (p < 0.05). While, GFAP IOD was significantly decreased in INS + VD (293.9 ± 36.6) groups, when compared to the VD group (p < 0.05) (As shown in  < 0.05) .p > 0.05). The IODs of p-tau and tau were significantly increased in the VD group compared with that in the VD group (p < 0.05). And, compared to the VD group, a significantly lower IODs of p-tau and tau were detected in the INS + VD group (p < 0.05). In addition, there was no significant difference for IOD of Aβ1-42 between CK, Sham, VD, and INS + VD groups (p > 0.05) (1-42.There was no significant difference for IODs of p-tau and tau between CK and Sham groups ( > 0.05) , indicatFrmd7 and Mpdz genes were the most downregulated and upregulated mRNAs, with FCs of −6.59 and 1.03 respectively between the INS + VD and VD groups. The Adcy1 and Tapbp genes were the most downregulated and upregulated mRNAs, with FCs of −6.89 and 1.00, respectively between the VD and CK groups.In total, 43.17 Gb of data were obtained from the transcriptome analysis. The effective data of each sample were distributed between 6.67 and 6.94G. Q30 bases were found in 92.46–93.38% of the data, and the average GC content was 50.55%. DEGs from the three-treatment comparison (CK vs. VD vs. INS + VD) with a |fold change| ≥ 1 and FDR < 0.05  were obtItpr1, Htr5a, Atp2b3, Shank3, Trpc, Cxcr4, Frmd7, Mpdz, Adcy1, and Tapbp) were determined by qRT-PCR with three replicates , neuron projection (GO:0043005), plasma membrane part (GO:0044459), and other functional categories  Supplem. And 61 p = 5.321E-11), neuroactive ligand-receptor interaction , and calcium signaling pathway  were the top 3 significantly altered pathways between the INS + VD and VD groups (p = 0.0002), glutamatergic synapse , and axon guidance  were also found to be significantly altered pathways between the INS + VD and VD groups (p = 0.002), oxytocin signaling pathway , cAMP signaling pathway  were the top 3 significantly altered pathways between the VD and CK groups (p = 0.04), serotonergic synapse , and glutamatergic synapse  were found to be significantly altered pathways between the VD and CK groups  based on VIP values (p = 0.046) was found between the INS + VD and VD groups in negative mode  and 12 upregulated)   were detgulated) . In addigulated) , and invgulated) .The compound-reaction-enzyme-gene networks in VD vs. CK and INS + VD vs. VD comparisons are shown in In addition, the DEGs related to the integrated pathways including Spp1, Frmd7, Asb16 were significantly upregulated, and Adcy1, Mfsd4a, and Mettl11b were significantly downregulated in the VD group compared to the CK group. The differentially accumulated metabolites: sphinganine 1-phosphate, 2′-Deoxyguanosine and 3-Methylhistidine were significantly upregulated, and L-Valine, Thiosulfate, and Tranexamic acid were significantly downregulated in VD group compared with the CK group.Furthermore, the DEGs related to the integration pathways including Nkx2-1, Asb15, and Mettl11b were significantly upregulated and Ppm1j, Sfrp5, and Prss56 were significantly downregulated in the INS + VD group compared with the VD group. The differentially accumulated metabolites 3-Methoxybenzyl alcohol and 2-keto-D-gluconic acid were significantly upregulated and downregulated, respectively, in the INS + VD group, compared with the VD group.Insulin and its receptors exist widely in the central nervous system, among which important signal transduction components are selectively distributed in the cognition-related hippocampus and other regions, with special biological effects, and play an important role in the growth and development of neurons, regulation of brain neurotransmitter transmission, utilization and uptake of glucose in the brain, learning and memory D . The phaNeuroinflammation is one of the pathogenic mechanisms of VD . The proTau protein is a relatively sensitive marker of neuronal ischemic damage . Tau pro1-42 expression levels between the VD, CK, and INS + VD groups. This finding is inconsistent with the results of several previous studies  receptors is one of the important mechanisms . Genes related to Ca2+ signaling (Itpr1), glutamatergic synaptic (Shank3), and axon guidance (Trpc5 and Cxcr4) were significantly differentially expressed among the three groups. These results suggested that insulin may affect glutamate release and Ca2+ overload by altering the expression of Ca2+ signaling pathway, glutamatergic synaptic pathway, and axon guidance pathway-related genes, modulating hippocampal synaptic plasticity, reducing brain damage, and improving cognitive function in VD model rats.Apoptosis of neuronal cells caused by activation of EAA receptors is also an important cause of VD . When brunctions . Li X. Gchanisms . In the Moreover, we detected 1 and 16 DEMs in the INS + VD vs. VD and VD vs. CK comparisons in negative and positive modes, enriched in 2 and 17 KEGG pathways. Although, no common metabolite was detected between VD vs. CK and INS + VD vs. VD comparisons, we found that the DEMs between the INS + VD and VD groups in negative mode and the VD and CK groups in positive mode were enriched in the metabolic pathway (ko01100). The metabolic pathway included metabolites of 2-keto-D-gluconic acid, 3-methylhistidine, sphinganine 1-phosphate, 2′-deoxyguanosine, allantoic acid, and uracil, which indicate that these metabolites may act as epigenetically, play an important role and are involved in the pathogenesis of VD and can be used as novel therapeutic target compounds for VD therapy.In addition, the DEGs related to the integrated pathways suggested that the Spp1, Frmd7, Asb16, Adcy1, Mfsd4a, and Mettl11b genes may regulate the synthesis and function of metabolites such as sphinganine 1-phosphate, 2′-deoxyguanosine and 3-methylhistidine, L-valine, thiosulfate, and tranexamic acid. The Nkx2-1, Asb15, Mettl11b, Ppm1j, Sfrp5, and Prss56 genes may regulate the synthesis and function of metabolites such as 3-methoxybenzyl alcohol and 2-keto-D-gluconic acid. However, little is known about the function of these genes and the regulation of these metabolites. It is necessary to identify them with further experiments.In summary, our study demonstrated that intranasal spray insulin could effectively reduce tau and p-tau levels, inhibit astrocyte inflammation, reduce neuron apoptosis, and improve cognitive function in VD model rats. The combined transcriptome and metabolome analyses suggested that the potential mechanism of insulin against VD involved genes were enriched in the calcium signaling pathway, cAMP signaling pathway, axon guidance, and glutamatergic synapse. Moreover, metabolites involved in metabolic pathway might participate in the protective effects of insulin against VD.