Network Pharmacology Analysis Research Articles

Health Benefits of Antioxidant Bioactive Compounds in Ginger (Zingiber officinale) Leaves by Network Pharmacology Analysis Combined with Experimental Validation.

Network pharmacology is an ideal tool to explore the effects of therapeutic components derived from plants on human metabolic diseases that are linked to inflammation. This study investigated the antioxidant effects of ginger leaves (GLs) and predicted targets for antioxidant activity. Quantitative and free radical scavenging analyses were performed to detect the main bioactive compounds of GLs and evaluate their antioxidant activities. Chemical diversity and network pharmacology approaches were used to predict key antioxidant components of GLs and their molecular targets. Nine major bioactive compounds of GLs were quantified using an internal standard method, and the antioxidant activity was evaluated using the DPPH and ABTS free radical scavenging methods. We first built the compound-gene-pathways and protein-protein interaction networks of GLs-related antioxidant targets and then conducted gene ontology and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses. Molecular docking results show that astragalin, a compound isolated from GLs, had the highest level of connectivity in the compound-target network and was involved in inflammation-related biosynthesis by directly impacting cytokine gene expression and PTGS2 inhibition markers. These findings not only suggest that the compounds isolated from GLs can be developed as potential antioxidants, but also demonstrate the applicability of network pharmacology to assess the potential of foods for disease treatment.

Matrine inhibits invasion and migration of gallbladder cancer via regulating the PI3K/AKT signaling pathway.

Gallbladder cancer (GBC) is a common malignant cancer in the biliary system, which poses a serious threat to human health. It is urgent to explore ideal drugs for the treatment of GBC. Matrine is the main active ingredient of Sophora flavescentis, with a wide range of biological activities encompassing anti-inflammatory, antiviral, immunomodulatory, and anti-tumor. However, the underlying mechanism by which Matrine treats GBC is still unclear. The purpose of this study is to investigate the anti-tumor effects of Matrine on GBC in vivo and in vitro and to clarify the potential regulatory mechanisms. Here, we found that Matrine had a significant killing effect on GBC through CCK8 and flow cytometry, including arrest of cell cycle, inhibition of GBC cell, and induction of apoptosis. Further in vivo studiesconfirmed the inhibitory effect of Matrine on tumor growth in NOZ xenografted nude mouse. At the same time, Matrine also significantly suppressed the migration and invasion of GBC cells through scratch and Transwell experiments. In addition, by detecting the mRNA and protein levels of epithelial-mesenchymal transition (EMT) and matrix metalloproteinases, Matrine furtherly substantiated the inhibitory role on invasion and migration of GBC. From a mechanistic perspective, network pharmacology analysis suggests that the potential targets of Matrine in the treatment of GBC are enriched in the PI3K/AKT signaling pathway. Subsequently, Matrine effectively decreased the abundance of p-PI3K and p-AKT protein in vivo and in vitro. More importantly, PI3K activator (740 Y-P) antagonized the anti-tumor effect of Matrine, while PI3K inhibitor (LY294002) increased the sensitivity of Matrine for GBC. Based on the above findings, we conclude that Matrine inhibits the invasion and migration of GBC by regulating PI3K/AKT signaling pathway. Our results indicate the crucial role and regulatory mechanism of Matrine in suppressing the growth of GBC, which provides a theoretical basis for Matrine to be a candidate drug for the treatment and research of GBC.

Paeoniae Radix Alba effectively attenuates Polygonum multiflorum Thunb. -induced idiosyncratic liver injury by modulating M2 macrophage polarization

Objective: Polygonum multiflorum Thunb. (PM) is a commonly used tonic herb known to cause idiosyncratic drug-induced liver injury (IDILI). This study explored the detoxification effects and potential mechanisms of action of Paeoniae Radix Alba (PRA) on PM-induced IDILI. Methods: Network pharmacology analysis was utilized to predict the related targets of "PRA-PM-innate immunity.” A non-hepatotoxic lipopolysaccharide (LPS) and PM-induced IDILI model was used to evaluate the detoxification effects of PRA by measuring liver function indicators, pathological examinations, and macrophage-related factors. Bone marrow-derived macrophages (BMDMs) were stimulated with IL-4 to differentiate into M2 macrophages, and the effects of PM and PRA on M2 macrophage polarization were explored. Results: Target screening of "PRA-PM-innate immunity" identified 21 intersecting targets, most of which were closely associated with macrophage polarization. In rat models of IDILI induced by PM, the combined use of PRA significantly reduced the extent of liver damage and the levels of inflammatory factors, while promoting the expression of M2 macrophage-related factors such as IL-4, IL-10, Arg1, and CD206. In vitro, PM dose-dependently inhibited the expression of the Arg1 protein and M2 macrophage-related genes, whereas PRA exhibited the opposite effect. When used in combination, PRA ameliorated the inhibitory effect of PM on M2 macrophage polarization. Conclusions: Our results demonstrate that PRA has a therapeutic effect on PM-induced IDILI; its mechanism may involve alleviating liver injury by promoting M2 macrophage polarization, thus reducing the expression of inflammatory factors.

A peptide from yak ameliorates hypoxia-induced kidney injury by inhibiting inflammation and apoptosis via Nrf2 pathway

Chronic kidney disease is a prevalent and severe significant complication of hypoxia. This study found that peptide LVYPFPGPIPN could protect hypoxia-induced renal injury in the animal model. Network pharmacology and molecular docking analysis indicated that cathepsin B (CTSB) and interleukin-1β (IL-1β) represent potential targets for the prevention/treatment of hypoxic-induced renal injury. GO analysis revealed the involvement of these genes in various biological processes, including apoptosis regulation, oxidative stress response, and adaptive immune modulation. Experimental results in vitro and in vivo demonstrated that peptide LVYPFPGPIPN could effectively inhibit apoptosis and stress responses of kidney cells by regulating the NRF2/IL-1β/mitochondrial apoptosis pathway, thereby protecting hypoxic human embryonic kidney cells from damage. The anti-hypoxic effect of the LVYPFPGPIPN offers a novel therapeutic clue for the treatment/prevention of hypoxic-induced kidney injury and inflammation-associated chronic kidney disease.

Study on the bioactive ingredients and mechanism of Huangqi against diabetic retinopathy based on network pharmacology and experimental verification.

Diabetic retinopathy (DR) is one of the most well-known microvascular complications of diabetes mellitus. As a traditional Chinese medicine, Huangqi (HQ), has been used for treating DR for a long time. However, its anti-DR active ingredients and mechanism are still unknown. Therefore, we designed this study to explore the active components and mechanism of HQ against DR via network pharmacology analysis. The ingredients of HQ, potential targets of HQ and DR were obtained from public databases. We employed the protein-protein interaction (PPI) network, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis to identify core targets and pathways of HQ against DR. Finally, molecular docking and vitro experiments were applied to validate our results. A total of 34 potential targets of HQ against DR were obtained. Based on PPI network, VEGFA, PTGS2, IL6 and CCL2 were considered as core targets. GO analysis involved 692 biological processes, 21 cellular components and 35 molecular functions. KEGG enrichment analysis manifested that the anti-DR effect of HQ was mainly mediated via the AGE-RAGE signaling pathway in diabetic complications. The molecular docking results indicated that kaempferol had higher affinity with CCL2, IL-6, VEGFA and PTGS2. The vitro experiments showed that the mRNA expressions of CCL2, IL-6, VEGFA and PTGS2 in ARPE-19 cell were differentially decreased after kaempferol treatment. This study preliminarily unveiled that the therapeutic efficacy of HQ against DR might be attributed to the reduced expression of CCL2, IL-6, VEGFA and PTGS2.

#971 Effects of arsenic trioxide on MMP9/IL-17A expression in lupus nephritis—results from bioinformatics analysis and in vitro studies

Abstract Background and Aims Previous research has indicated that ATO treatment at low doses may decrease the likelihood of flares in active systemic lupus erythematosus (SLE) patients, but the pharmacological mechanisms of such effect have not been studied. Method Machine learning and network pharmacology analysis were employed to explore the potential targets from differentially expressed genes (DEGs) of human SLE and LN peripheral blood mononuclear cells (PBMCs) microarray datasets and pharmacological databases. GO and KEGG analyses were performed, and the protein-protein interaction (PPI) network was constructed. The association of these characteristic genes and immune cells were further examined and validated. Results Three machine learning models (RF, SVM, and XGB) were utilized to identify five immunoregulatory genes in SLE among twelve intersection DEGs. Among these genes, MMP9 exhibited the highest ROC AUC (0.92) in predicting disease development. KEGG analysis revealed significant associations with the IL-17 signaling pathway (p = 1.67E-18). Furthermore, ssGSEA analysis indicated positive correlations between MMP9 and macrophages (correlation coefficient 0.88) as well as neutrophils (correlation coefficient 0.66). In vitro treatment of PBMCs from lupus nephritis (LN) patients with ATO resulted in downregulation of MMP9 and IL-17A, validating our bioinformatics findings. Conclusion ATO has the potential to mitigate the MMP/IL-17A pathways in PBMCs in LN patients, and hence maybe a promising and innovative therapeutic alternative.

Analysis of an Aqueous Extract from Turkish Galls Based on Multicomponent Qualitative and Quantitative Analysis Combined with Network Pharmacology and Chemometric Analysis.

The current quality control method for Turkish gall (TG) is limited to assessing total tannin or gallic acid (GA), which offers a basic level of quality control but does not fully capture the true quality of TG. Therefore, it is essential to establish a comprehensive method that utilizes multiple indicators to assess the intrinsic quality of TG. This research utilized UPLC-Q-TOF-MS/MS technology to qualitatively analyze the chemical composition of TG. Subsequently, the potential main active ingredients, targets, and pathways of TG in treating recurrent aphthous ulcers (RAU) were explored and analyzed using network pharmacology technology. Quantitative analysis of multicomponents by single marker (QAMS) was then employed to quantify the primary pharmacodynamic components in TG. Finally, chemometrics analysis was utilized to interpret the measured results and identify the markers of scavenging quality. The study identified 36 chemical components in TG, highlighting ellagic acid (EA), GA, and so on as key components in treating RAU. A method for simultaneously determining GA, EA, 1,2,3,6-tetra-O-galloyl-β-D-glucose (TEGG) and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PEGG) in TG was established. Statistical analysis revealed significant differences in the content of these 4 components across 14 batches of TG, with GA and PEGG identified as the primary contributors to the variations. This study determined a quality index for TG, providing a reference for quality evaluation and introducing a cost-effective and efficient quality control method. Furthermore, it addressed the challenge of developing new Chinese medicine by overcoming the lack of reference substances.

Exploring the mechanism of 6-Methoxydihydrosanguinarine in the treatment of lung adenocarcinoma based on network pharmacology, molecular docking and experimental investigation

Background6-Methoxydihydrosanguinarine (6-MDS) has shown promising potential in fighting against a variety of malignancies. Yet, its anti‑lung adenocarcinoma (LUAD) effect and the underlying mechanism remain largely unexplored. This study sought to explore the targets and the probable mechanism of 6-MDS in LUAD through network pharmacology and experimental validation.MethodsThe proliferative activity of human LUAD cell line A549 was evaluated by Cell Counting Kit-8 (CCK8) assay. LUAD related targets, potential targets of 6-MDS were obtained from databases. Venn plot analysis were performed on 6-MDS target genes and LUAD related genes to obtain potential target genes for 6-MDS treatment of LUAD. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was utilized to perform a protein-protein interaction (PPI) analysis, which was then visualized by Cytoscape. The hub genes in the network were singled out by CytoHubba. Metascape was employed for GO and KEGG enrichment analyses. molecular docking was carried out using AutoDock Vina 4.2 software. Gene expression levels, overall survival of hub genes were validated by the GEPIA database. Protein expression levels, promotor methylation levels of hub genes were confirmed by the UALCAN database. Timer database was used for evaluating the association between the expression of hub genes and the abundance of infiltrating immune cells. Furthermore, correlation analysis of hub genes expression with immune subtypes of LUAD were performed by using the TISIDB database. Finally, the results of network pharmacology analysis were validated by qPCR.ResultsExperiments in vitro revealed that 6-MDS significantly reduced tumor growth. A total of 33 potential targets of 6-MDS in LUAD were obtained by crossing the LUAD related targets with 6-MDS targets. Utilizing CytoHubba, a network analysis tool, the top 10 genes with the highest centrality measures were pinpointed, including MMP9, CDK1, TYMS, CCNA2, ERBB2, CHEK1, KIF11, AURKB, PLK1 and TTK. Analysis of KEGG enrichment hinted that these 10 hub genes were located in the cell cycle signaling pathway, suggesting that 6-MDS may mainly inhibit the occurrence of LUAD by affecting the cell cycle. Molecular docking analysis revealed that the binding energies between 6-MDS and the hub proteins were all higher than − 6 kcal/Mol with the exception of AURKB, indicating that the 9 targets had strong binding ability with 6-MDS.These results were corroborated through assessments of mRNA expression levels, protein expression levels, overall survival analysis, promotor methylation level, immune subtypes andimmune infiltration. Furthermore, qPCR results indicated that 6-MDS can significantly decreased the mRNA levels of CDK1, CHEK1, KIF11, PLK1 and TTK.ConclusionsAccording to our findings, it appears that 6-MDS could possibly serve as a promising option for the treatment of LUAD. Further investigations in live animal models are necessary to confirm its potential in fighting cancer and to delve into the mechanisms at play.

Untargeted serum and gastric metabolomics and network pharmacology analysis reveal the superior efficacy of zingiberis rhizoma recens-/euodiae fructus-processed Coptidis Rhizoma on gastric ulcer rats

Ethnopharmacological relevanceZingiberis rhizoma recens-/wine-/euodiae fructus-processed Coptidis Rhizoma (CR, zCR/wCR/eCR) are the commonly used processed products of CR in clinic. After being processed with different excipients, the efficacy of CR will change accordingly. I.e., wCR could resolve excessive heat of the upper energizer, zCR could eliminate gastric heat and harmonize the stomach, eCR could smooth the liver and harmonize the stomach. However, the underlying mechanisms were still unclear. Aim of the studyTo further verify the differential efficacy of the three processed CR products and compare the mechanisms on gastric ulcer. Material and methodsFirst, a GU model, whose onset is closely related to the heat in stomach and the disharmony between liver and stomach, was established, and the therapeutic effects of zCR/wCR/eCR/CR were evaluated by pathologic observation and measurement of cytokine levels. Second, metabolomics analysis and network pharmacology were conducted to reveal the differential intervening mechanism of zCR/eCR on GU. Third, the predicted mechanisms from metabolomics analysis and network pharmacology were validated using western blotting, flow cytometry and immunofluorescence. ResultszCR/wCR/eCR/CR could alleviate the pathologic damage to varying degrees. In metabolomics research, fewer metabolic pathways were enriched in serum samples, and most of them were also present in the results of gastric tissue samples. The gastroprotective, anti-inflammatory, antioxidant, and anti-apoptotic effects of zCR/wCR/eCR/CR might be due to their interference on histidine, arachidonic acid, and glycerophospholipids metabolism. Quantitative results indicated that zCR/eCR had a better therapeutic effect than wCR/CR in treating GU. A comprehensive analysis of metabolomics and network pharmacology revealed that zCR and eCR exerted anti-GU effects via intervening in five core targets, including AKT, TNF, IL6, IL1B and PPARG. In the validation experiment, zCR/eCR could significantly reverse the abnormal expression of proteins related to apoptosis, inflammation, oxidative stress, gastric function, as well as the PI3K/AKT signaling pathways. ConclusionzCR and eCR could offer gastroprotective benefits by resisting inflammation and apoptosis, inhibiting gastric-acid secretion, as well as strengthening gastric mucosal defense and antioxidant capacity. Integrating network pharmacology and metabolomics analysis could reveal the acting mechanism of drugs and promote the development of medications to counteract GU.

Integrating metabolomics and network pharmacology analysis to explore mechanism of Pueraria lobata against pulmonary fibrosis: Involvement of arginine metabolism pathway

Ethnopharmacological relevancePueraria lobata (Willd.) Ohwi is a typical medicinal and edible plant with a long application history in China and Southeast Asia. As a widely used traditional medicine, P. lobata exhibits the properties of anti-inflammatory, antipyretic, antioxidant, relieving cough and asthma. Particularly, the increasing evidence indicates that the P. lobata has the therapeutic effect on fibrotic-related diseases in terms of metabolic regulation. However, the mechanisms of P. lobata on pulmonary fibrosis (PF) has not been thoroughly explored. Aim of the studyThis study aimed to explore the effect of arginine metabolites of P. lobata against PF model by integrating metabolomics and network pharmacology analysis. It might provide a new idea for the target finding of P. lobata anti-pulmonary fibrosis. Materials and methodsIn this study, the Sprague Dawley (SD) rats were randomly divided into five experimental groups: saline-treated control group, bleomycin-induced fibrosis group, prednisolone acetate group, P. lobata 3.2 g/kg group and P. lobata 6.4 g/kg group. The therapeutic effect of P. lobata on bleomycin-induced PF in rats was evaluated by clinical symptoms such as lung function, body weight, hematoxylin eosin staining (HE), Masson staining and hydroxyproline assay. Next, the plasma metabolomics analysis was carried out by LC-MS to explore the pathological differences between the group of control, PF and P. lobata-treated rats. Then, the network pharmacology study coupled with experimental validation was conducted to analysis the results of metabolic research. We constructed the “component-target-disease” network of P. lobata in the treatment of PF. In addition, the molecular docking method was used to verify the interaction between potential active ingredients and core targets of P. lobata. Finally, we tested NOS2 and L-OT in arginine-related metabolic pathway in plasma of the rats by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was performed to observe the level of TNF-α mRNA and MMP9 mRNA. And we tested the expression of TNF-α and MMP9 by Western blot analysis. ResultsOur findings revealed that P. lobata improved lung function and ameliorated the pathological symptoms, such as pathological damage, collagen deposition, and body weight loss in PF rats. Otherwise, the plasma metabolomics were employed to screen the differential metabolites of amino acids, lipids, flavonoids, arachidonic acid metabolites, glycoside, etc. Finally, we found that the arginine metabolism signaling mainly involved in the regulating of P. lobata on the treatment of PF rats. Furtherly, the network pharmacology predicted that the arginine metabolism pathway was contained in the top 20 pathways. Next, we integrated metabolomics and network pharmacology that identified NOS2, MMP9 and TNF-α as the P. lobata regulated hub genes by molecular docking. Importantly, it indicated a strong affinity between the puerarin and the NOS2. P. lobata attenuated TNF-α, MMP-9 and NOS2 levels, suppressed TNF-α and MMP-9 protein expression, and decreased L-OT and NOS2 content in PF rats. These results indicated that the effects of P. lobata may ameliorated PF via the arginine metabolism pathway in rats. Therefore, P. lobata may be a potential therapeutic agent to ameliorated PF. ConclusionIn this work, we used metabolomics and network pharmacology to explore the mechanisms of P. lobata in the treatment of PF. Finally, we confirmed that P. lobata alleviated BLM-induced PF in rats by regulating arginine metabolism pathway based on reducing the L-OT and NOS2-related signal molecular. The search for the biomarkers finding of arginine metabolism pathway revealed a new strategy for P. lobata in the treatment of PF.

Exploration of the mechanism of Traditional Chinese Medicine for anxiety and depression in patients with diarrheal irritable bowel syndrome based on network pharmacology and meta-analysis.

The efficacy of Chinese herbal medicine (CHM) in managing irritable bowel syndrome with diarrhea (IBS-D) accompanied by anxiety and depression remains uncertain. Thus, a systematic review was carried out employing meta-analysis and network pharmacology to ascertain the efficacy and underlying mechanisms of CHM therapy. By conducting a systematic review, including literature search, screening, and data extraction, we identified 25 randomized controlled trials to assess CHM's effectiveness in treating irritable bowel syndrome alongside anxiety and depression. Network pharmacology was utilized to scrutinize the metabolite utility of CHM in addressing this condition. Potential primary mechanisms were synthesized using information sourced from the PubMed database. Twenty-five studies, including 2055 patients, were analyzed, revealing significant treatment efficacy for IBS-D in the trial group compared to controls [OR = 4.01, 95% CI (2.99, 5.36), I2 = 0%] Additionally, treatment for depression [SMD = -1.08, 95% CI (-1.30, -0.86), p < 0.00001, I2 = 68%; SDS: SMD = -1.69, 95% CI (-2.48, -0.90), p < 0.0001, I2 = 96%] and anxiety [HAMA: SMD = -1.29, 95% CI (-1.68, -0.91), p < 0.00001, I2 = 89%; SAS: SMD = -1.75, 95% CI (-2.55, -0.95), p < 0.00001, I2 = 96%] significantly improved in the trial group. Furthermore, the trial group exhibited a significantly lower disease relapse rate [OR = 0.30, 95% CI (0.20, 0.44), p < 0.00001, I2 = 0%]. CHM treatment consistently improved IBS severity (IBS-SSS) and symptom scores. Network pharmacology analysis identified key chemical metabolites in traditional Chinese medicine formulations, including Beta-sitosterol, Stigmasterol, Quercetin, Naringenin, Luteolin, Kaempferol, Nobiletin, Wogonin, Formononetin, and Isorhamnetin. Utilizing the STRING database and Cytoscape v3.9.0 software, a protein-protein interaction (PPI) network revealed the top eight key targets: IL-6, TNF, PPARG, PTGS2, ESR1, NOS3, MAPK8, and AKT1, implicated in anti-inflammatory responses, antioxidant stress modulation, and neurotransmitter homeostasis maintenance. Chinese Herbal Medicine (CHM) offers a promising and safe treatment approach for patients dealing with Diarrheal Irritable Bowel Syndrome (IBS-D) accompanied by anxiety and depression; thus, indicating its potential for practical implementation. The most active metabolites of CHM could simultaneously act on the pathological targets of IBS-D, anxiety, and depression.The diverse scope of CHM's therapeutic role includes various aspects and objectives, underscoring its potential for broad utilization.

Network pharmacology integrated with experimental validation to elucidate the mechanisms of action of the Guizhi-Gancao Decoction in the treatment of phenylephrine-induced cardiac hypertrophy

Context The mechanisms of Traditional Chinese Medicine (TCM) Guizhi-Gancao Decoction (GGD) remain unknown. Objective This study explores the mechanisms of GGD against cardiac hypertrophy. Materials and methods Network pharmacology analysis was carried out to identify the potential targets of GGD. In vivo experiments, C57BL/6J mice were divided into Con, phenylephrine (PE, 10 mg/kg/d), 2-chloroadenosine (CADO, the stable analogue of adenosine, 2 mg/kg/d), GGD (5.4 g/kg/d) and GGD (5.4 g/kg/d) + CGS15943 (a nonselective adenosine receptor antagonist, 4 mg/kg/d). In vitro experiments, primary neonatal rat cardiomyocytes (NRCM) were divided into Con, PE (100 µM), CADO (5 µM), GGD (10−5 g/mL) and GGD (10−5 g/mL) + CGS15943 (5 µM). Ultrasound, H&E and Masson staining, hypertrophic genes expression and cell surface area were conducted to verify the GGD efficacy. Adenosine receptors (ADORs) expression were tested via real-time polymerase chain reaction (PCR), western blotting and immunofluorescence analysis. Results Network pharmacology identified ADORs among those of the core targets of GGD. In vitro experiments demonstrated that GGD attenuated PE-induced increased surface area (with an EC50 of 5.484 × 10−6 g/mL). In vivo data shown that GGD attenuated PE-induced ventricular wall thickening. In vitro and in vivo data indicated that GGD alleviated PE-induced hypertrophic gene expression (e.g., ANP, BNP and MYH7/MYH6), A1AR over-expression and A2aAR down-expression. Moreover, CADO exerts effects similar to GGD, whereas CGS15943 eliminated most effects of GGD. Discussion and conclusions Our findings suggest the mechanism by which GGD inhibits cardiac hypertrophy, highlighting regulation of ADORs as a potential therapeutic strategy for HF.

Quercetin, Main Active Ingredient of Moutan Cortex, Alleviates Chronic Orofacial Pain via Block of Voltage-Gated Sodium Channel.

Chronic orofacial pain (COP) therapy is challenging, as current medical treatments are extremely lacking. Moutan Cortex (MC) is a traditional Chinese medicine herb widely used for chronic inflammatory diseases. However, the mechanism behind MC in COP therapy has not been well-established. The purpose of this study was to identify the active ingredients of MC and their specific underlying mechanisms in COP treatment. In this study, the main active ingredients and compound-target network of MC in COP therapy were identified through network pharmacology and bioinformatics analysis. Adult male Sprague-Dawley rats received oral mucosa lipopolysaccharide (LPS) injection to induce COP. Pain behaviors were evaluated by orofacial mechanical nociceptive assessment after intraganglionar injection. In vitro inflammatory cytokines in LPS-pretreated human periodontal ligament stem cells (hPDLSCs) and rat primary cultural trigeminal ganglion (TG) neurons were quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Schrödinger software was used to verify the molecular docking of quercetin and critical targets. Whole-cell recording electrophysiology was used to evaluate the effect of quercetin on voltage-gated sodium (Na v ) channel in rat TG neurons. The assembled compound-target network consisted of 4 compounds and 46 targets. As 1 of the active components of MC correlated with most related targets, quercetin alleviated mechanical allodynia in LPS-induced rat model of COP (mechanical allodynia threshold median [interquartile range (IQR) 0.5 hours after drug administration: vehicle 1.3 [0.6-2.0] g vs quercetin 7.0 [6.0-8.5] g, P = .002). Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that immune response and membrane functions play essential roles in MC-COP therapy. Five of the related targets were identified as core targets by protein-protein interaction analysis. Quercetin exerted an analgesic effect, possibly through blocking Na v channel in TG sensory neurons (peak current density median [IQR]: LPS -850.2 [-983.6 to -660.7] mV vs LPS + quercetin -589.6 [-711.0 to -147.8] mV, P = .006) while downregulating the expression level of proinflammatory cytokines-FOS (normalized messenger RNA [mRNA] level mean ± standard error of mean [SEM]: LPS [2. 22 ± 0.33] vs LPS + quercetin [1. 33 ± 0.14], P = .034) and TNF-α (normalized mRNA level mean ± SEM: LPS [8. 93 ± 0.78] vs LPS + quercetin [3. 77 ± 0.49], P < .0001). Identifying Na v as the molecular target of quercetin clarifies the analgesic mechanism of MC, and provides ideas for the development of novel selective and efficient chronic pain relievers.

Combination of UPLC-Q-TOF/MS and network pharmacology to reveal the mechanism of Chaihu-jia-Longgu-Muli decoction for treating vertigo with anxiety disorder.

Chaihu-jia-Longgu-Muli decoction (CLMD) has been proven clinically effective in treating vertigo with anxiety disorder. However, the mechanism is not clear. This study aimed to explore the mechanism of CLMD in treating vertigo with anxiety disorder based on ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) and network pharmacology. UPLC-Q-TOF/MS was performed to identify the compounds in blood and the targets of compounds of CLMD in vertigo and anxiety were searched using databases. A protein-protein interaction network was built to screen the core targets. The core targets were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. In addition, the vertigo with anxiety rat model was used to verify the results. A total of 22 compounds were absorbed into the blood. Eighty-one potential targets associated with CLMD for vertigo with anxiety disorder were identified through network pharmacological analysis. Subsequently, GO and KEGG analysis showed that CLMD treatment for vertigo with anxiety disorder is associated with neurotransmitter levels and other pertinent physiological processes. The results of the animal experiments showed that CLMD decreased the levels of serotonin, norepinephrine and dopamine, alleviating the symptoms of vertigo and anxiety disorder in model rats. The study revealed CLMD could alleviate vertigo and anxiety symptoms through reducing the levels of neurotransmitters.

Nardosinone relieves metabolic-associated fatty liver disease and promotes energy metabolism through targeting CYP2D6

BackgroundNardosinone, a major extract of Rhizoma nardostachyos, plays a vital role in sedation, neural stem cell proliferation, and protection of the heart muscle. However, the huge potential of nardosinone in regulating lipid metabolism and gut microbiota has not been reported, and its potential mechanism has not been studied. PurposeTo explore the regulation of nardosinone on liver lipid metabolism and gut microbiota. MethodsIn this study, the role of nardosinone in lipid metabolism was investigated in vitro and in vivo by adding it to mouse feed and HepG2 cell culture medium. And 16S rRNA gene sequencing was used to explore its regulatory effect on gut microbiota. ResultsResults showed that nardosinone could improve HFD-induced liver injury and abnormal lipid metabolism by promoting mitochondrial energy metabolism in hepatocytes, alleviating oxidative stress damage, and regulating the composition of the gut microbiota. Mechanistically, combined with network pharmacology and reverse docking analysis, it was predicted that CYP2D6 was the target of nardosinone, and the binding was verified by cellular thermal shift assay (CETSA). ConclusionsThis study highlights a novel mechanism function of nardosinone in regulating lipid metabolism and gut microbiota. It also predicts and validates CYP2D6 as a previously unknown regulatory target, which provides new possibilities for the application of nardosinone and the treatment of metabolic-associated fatty liver disease.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

Molecular Mechanism of Yangshen Maidong Decoction in the Treatment of Chronic Heart Failure based on Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulations.

Chronic heart failure (CHF) is a complex multifactorial clinical syndrome leading to abnormal cardiac structure and function. The severe form of this ailment is characterized by high disability, high mortality, and morbidity. Worldwide, 2-17% of patients die at first admission, of which 17-45% die within 1 year of admission and >50% within 5 years. Yangshen Maidong Decoction (YSMDD) is frequently used to treat the deficiency and pain of the heart. The specific mechanism of action of YSMDD in treating CHF, however, remains unclear. Therefore, a network pharmacology-based strategy combined with molecular docking and molecular dynamics simulations was employed to investigate the potential molecular mechanism of YSMDD against CHF. The effective components and their targets of YSMDD and related targets of CHF were predicted and screened based on the public database. The network pharmacology was used to explore the potential targets and possible pathways that involved in YSMDD treated CHF. Molecular docking and molecular dynamics simulations were performed to elucidate the binding affinity between the YSMDD and CHF targets. Screen results, 10 main active ingredients, and 6 key targets were acquired through network pharmacology analysis. Pathway enrichment analysis showed that intersectional targets associated pathways were enriched in the Prostate cancer pathway, Hepatitis B pathway, and C-type lectin receptor signaling pathways. Molecular docking and molecular dynamics simulations analysis suggested 5 critical active ingredients have high binding affinity to the 5 key targets. This research shows the multiple active components and molecular mechanisms of YSMDD in the treatment of CHF and offers resources and suggestions for future studies.

Network Pharmacology, Molecular Docking, and Experimental Validation on Guiluoshi Anzang Decoction Against Premature Ovarian Insufficiency.

Premature Ovarian Insufficiency (POI) is a disease suffered by women under the age of 40 when ovarian function has declined, seriously affecting both the physical and mental health of women. Guiluoshi Anzang decoction (GLSAZD) has been used for a long time and has a unique therapeutic effect on improving ovarian function. This study aims to investigate the mechanism of GLSAZD in treating POI through network pharmacology, molecular docking, and experimental verification. In this study, the active ingredients of Guiluoshi Anzang Decoction and the targets of POI were obtained from TCMSP, BATMANN-TCM, Uniprot, GeneCards, and other databases, and network pharmacology analysis was performed. Molecular docking was conducted to validate the affinity of the main active ingredient of GLSAZD to key POI targets. A POI SD rat model was established, and HE staining, ELISA, Real-time PCR, and Western blot experiments were performed to verify the predicted core targets and the therapeutic effects. 10 core targets and the top 5 ingredients were screened out. Molecular docking showed core targets AKT1, CASP3, TNF, TP53, and IL6 had stable binding with the core 5 ingredients quercetin, kaempferol, beta-sitosterol, luteolin, and Stigmasterol. GO and KEGG enrichment analysis demonstrated the mechanism involved in the positive regulation of gene expression, PI3K-AKT signaling pathway, and apoptosis signaling pathways. Animal experiments indicated GLSAZD could up-regulate the protein expression of p-PI3K and p-AKT1 and the mRNA expression of STAT3 and VEGF, down-regulate TP53 and Cleaved Caspase-3 protein expression in rat`s ovarian tissues and serum TNF-α and IL-6 protein levels, activate PI3K-AKT signaling pathway and inhibit the apoptosis signaling pathway. GLSAZD treats POI through multi-component, multi-target, and multi-pathway approaches. This study provided evidence for its clinical application in treating POI and shed light on the study of traditional medicine of the Guangxi Zhuang Autonomous Region in China.

Quality markers of Polygala fallax Hemsl decoction based on qualitative and quantitative analysis combined with network pharmacology and chemometric analysis.

Polygala fallax Hemsl (PFH) is a widely used herbal medicine in Guangxi, China. At present, research on PFH mainly focuses on extraction technology and cultivation, lacking quality control standards for systematic evaluation. The study aimed to assess the quality of PFH from different sources and to predict markers that would help assess quality. Fingerprinting of 15 batches of PFH samples was performed by ultra-high performance liquid chromatography (UPLC) and similarity was assessed using hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares discrimination (OPLS-DA). Differential components were screened by mathematical analysis, and a "component-target-pathway" network map was constructed in combination with network pharmacology, quality markers (Q-markers) of PFH were predicted, and quantitative analysis was performed. Fifteen batches were fingerprinted for PFH, with 11 common peaks, and peak 5 was identified as 4-hydroxybenzoic acid, which was generally consistent with the results of HCA, PCA, and OPLS-DA. Network pharmacology screened 18 potential compounds, 45 core targets, and 20 key pathways, integrating fingerprinting, pattern recognition, and network pharmacology methods. One of the potential Q-markers that can identify the principle of testability, efficacy, and specificity is 4-hydroxybenzoic acid, whose content ranges from 0.0188 to 1.4517 mg/g. The potential Q-markers of PFH were predicted by integrating fingerprinting, pattern recognition, and network pharmacological analysis, which provided a scientific basis for the overall control and evaluation of the quality of PFH and a theoretical reference for the study of the quality standard of multi-base traditional Chinese medicine.

Phlorizin from Lithocarpus litseifolius [Hance] Chun ameliorates FFA-induced insulin resistance by regulating AMPK/PI3K/AKT signaling pathway

BackgroundInsulin resistance (IR) is the central pathophysiological feature in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes mellitus (T2DM), hypertension, and dyslipidemia. As the main active ingredient in Lithocarpus litseifolius [Hance] Chun, previous studies have shown that phlorizin (PHZ) can reduce insulin resistance in the liver. However, the effect of phlorizin on attenuating hepatic insulin resistance has not been fully investigated, and whether this effect is related to AMPK remains unclear. PurposeThe present study aimed to further investigate the effect of phlorizin on attenuating insulin resistance and the potential action mechanism. MethodsFree fatty acids (FFA) were used to induce insulin resistance in HepG2 cells. The effects of phlorizin and FFA on cell viability were detected by MTT analysis. Glucose consumption, glycogen synthesis, intracellular malondialdehyde (MDA), superoxide dismutase (SOD), total cholesterol (TC), and triglyceride (TG) contents were quantified after phlorizin treatment. Glucose uptake and reactive oxygen species (ROS) levels in HepG2 cells were assayed by flow cytometry. Potential targets and signaling pathways for attenuating insulin resistance by phlorizin were predicted by network pharmacological analysis. Moreover, the expression levels of proteins related to the AMPK/PI3K/AKT signaling pathway were detected by western blot. ResultsInsulin resistance was successfully induced in HepG2 cells by co-treatment of 1 mM sodium oleate (OA) and 0.5 mM sodium palmitate (PA) for 24 h. Treatment with phlorizin promoted glucose consumption, glucose uptake, and glycogen synthesis and inhibited gluconeogenesis in IR-HepG2 cells. In addition, phlorizin inhibited oxidative stress and lipid accumulation in IR-HepG2 cells. Network pharmacological analysis showed that AKT1 was the active target of phlorizin, and the PI3K/AKT signaling pathway may be the potential action mechanism of phlorizin. Furthermore, western blot results showed that phlorizin ameliorated FFA-induced insulin resistance by activating the AMPK/PI3K/AKT signaling pathway. ConclusionPhlorizin inhibited oxidative stress and lipid accumulation in IR-HepG2 cells and ameliorated hepatic insulin resistance by activating the AMPK/PI3K/AKT signaling pathway. Our study proved that phlorizin played a role in alleviating hepatic insulin resistance by activating AMPK, which provided experimental evidence for the use of phlorizin as a potential drug to improve insulin resistance.