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ObjectiveTo investigate the effect of Ganoderma lucidum polysaccharides （GLP） on the proliferation， migration， cycle， and apoptosis of hepatocellular carcinoma SKHEP1 and Huh7 cells and to explore the underlying mechanism. MethodSK-HEP-1 and Huh-7 cells were classified into the blank group and low-， medium-， and high-dose GLP groups （3.5， 7， 14 g·L-1）. The proliferation of the cells was examined by cell counting kit-8 （CCK8） assay， and the migration by scratch assay. Cell cycle was measured by flow cytometry and apoptosis was detected based on Hoechst33258 staining. In addition， the expression of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated PI3K （pPI3K）， and phosphorylated Akt （pAkt） in the cells was determined by Western blot. ResultCompared with the blank group， the three doses of GLP reduced the proliferation and migration of SKHEP1 and Huh7 cells （P<0.05）， increased the percentage of cells in G1 phase （P<0.05）， and decreased percentage of cells in S and G2 phase （P<0.05）. In addition， the three doses can induce apoptosis of both SK-HEP-1 and Huh-7 cells， particularly the high dose. Moreover， the three doses of GLP lowered the levels of pPI3K and pAkt （P<0.05）. ConclusionGLP significantly inhibited the malignant phenotype of SK-HEP-1 and Huh-7 cells through the PI3K/Akt signaling pathway.
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The present study aimed to explore the main active components and potential mechanisms of Panax notoginseng saponins(PNS) and osteopractic total flavone(OTF) in the treatment of osteoporosis(OP) through network pharmacology, molecular docking and in vitro cell experiments, which was expected to provide a theoretical basis for clinical applications. The blood-entering components of PNS and OTF were obtained from literature search and online database, and their potential targets were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The OP targets were obtained by means of searching Online Mendelian Inheritance in Man(OMIM) and GeneCards. The common targets of the drug and disease were screened by Venn. Cytoscape was used to construct a "drug-component-target-disease" network, and the core components were screened according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened according to the node degree. GO and KEGG enrichment analysis of potential therapeutic targets were carried out by R language. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock Vina. Finally, HIF-1 signaling pathway was selected for in vitro experimental verification according to the results of KEGG pathway analysis. Network pharmacology showed that there were 45 active components such as leachianone A, kurarinone, 20(R)-protopanaxatriol, 20(S)-protopanaxatriol, and kaempferol, and 103 therapeutic targets such as IL6, AKT1, TNF, VEGFA and MAPK3 involved. PI3K-AKT, HIF-1, TNF and other signaling pathways were enriched. Molecular docking revealed that the core components had good binding ability to the core targets. In vitro experiments found that PNS-OTF could up-regulate the mRNA expression levels of HIF-1α, VEGFA and Runx2, indicating that the mechanism of PNS-OTF in treating OP may be related to the activation of HIF-1 signaling pathway, and thus PNS-OTF played a role in promoting angiogenesis and osteogenic differentiation. In conclusion, this study predicted the core targets and pathways of PNS-OTF in treating OP based on network pharmacology and carried out in vitro experimental verification, which reflected the characteristics of multi-component, multi-target and multi-pathway synergy of PNS-OTF, and provided new ideas for the future clinical treatment of OP.
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Humans , Molecular Docking Simulation , Network Pharmacology , Osteogenesis , Phosphatidylinositol 3-Kinases , Osteoporosis , Databases, GeneticABSTRACT
Objective:To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment.Methods:Experimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2' ,7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups.Results:In vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups ( F=43.478, 36.643, 24.464; P<0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group ( F=32.598, P<0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference ( F=47.267, P<0.01). The mRNA ( F=51.563, 32.192, 44.473; P<0.01) and protein levels ( F=63.372, 54.463, 48.412; P<0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. Conclusion:Met can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.
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Objective To investigate the protective effects of the total bakkenolides from P.tricholobus on improving hypoxia tolerance in mice. Methods Mice normobaric pressure hypoxia model and oxygen glucose deprivation model in PC12 cells were established, and the effects of PTB on survival time, serum lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content, brain and heart superoxide dismutase (SOD) and reduced glutathione (GSH) activities, brain tissue pathological changes and cell survival were observed. Results The total bakkenolides from P.tricholobus had prolonged the survival time of mice in confined spaces, increased the activity of SOD and GSH, reduced the production of lipid peroxidation, decreased the degree of anaerobic glycolysis, protected the structure and function of neural cells, and improved the survival rate of OGD-treated cells. Conclusion The total bakkenolides from P.tricholobus could promote the hypoxia tolerance in mice which might be related to scavenging oxygen free radicals, inhibiting lipid peroxidation reaction and protecting the structures and functions of nerve cells.
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ObjectiveTo predict the potential targets and possible related signaling pathways of Salviae Miltiorrhizae Radix et Rhizoma against bladder cancer （BC） based on network pharmacology and verify the potential molecular mechanism through in vitro cell experiment. MethodActive components of Salviae Miltiorrhizae Radix et Rhizoma were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and BC-related targets were searched from GeneCards and Online Mendelian Inheritance in Man （OMIM）. Via Venny2.1， the potential targets of Salviae Miltiorrhizae Radix et Rhizoma against BC were screened out and the Venn diagram was plotted. Protein-protein interaction （PPI） network was constructed by STRING， followed by Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Gnomes （KEGG） pathway enrichment with DAVID. Cell Counting Kit-8 （CCK-8） assay was employed to detect the inhibitory effect of tanshinone ⅡA （Tan ⅡA）， cryptotanshinone （CPT）， and luteolin （LUT） at different concentration （0， 1， 2， 4， 8， 16， 32 μmol·L-1） on the proliferation of BC T24 and 5637 cells， propidium iodide （PI） staining to analyze the apoptosis of 5637 cells induced by Tan ⅡA， CPT， and LUT （0， 4， 8 μmol·L-1）， and Western blotting to detect the regulatory effect of Tan ⅡA （0， 4， 8， 16 μmol·L-1） on the expression of key target proteins. ResultA total of 65 active components and 39 anti-BC targets of Salviae Miltiorrhizae Radix et Rhizoma were screened out. The anti-BC targets were mainly involved in the KEGG pathways of neuron-ligand-receptor interaction， phosphatidylinositol 3-kinases （PI3K）/protein kinase B （Akt） signaling pathway， epidermal growth factor receptor （EGFR） tyrosine kinase inhibitor resistance， and hypoxia inducible factor （HIF）-1 signaling pathway. As for the CCK-8 assay， compared with the blank group， Tan ⅡA， CPT， and LUT significantly inhibited the proliferation of T24 and 5637 cells， particularly the 5637 cells. The half maximal inhibitory concentration （IC50） of Tan ⅡA on 5637 cells was significantly lower than that of CPT and LUT. Moreover， compared with the blank group， Tan ⅡA， CPT， and LUT all induced the apoptosis of 5637 cells， and the effect followed the order of Tan ⅡA>CPT>LUT （P<0.05）. Western blot showed that Tan ⅡA significantly reduced the expression of EGFR， p-PI3K， and p-Akt in 5637 cells in a concentration-dependent manner compared with the blank group （P<0.05）. ConclusionSalviae Miltiorrhizae Radix et Rhizoma exerts therapeutic effect on BC through multiple components， multiple targets， and multiple pathways. The mechanism is the likelihood that it down-regulates the expression of EGFR， p-PI3K， and p-Akt proteins， thus further inhibits cell proliferation， and induces apoptosis.
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ObjectiveTo investigate the potential pharmacological mechanism of Xinmaikang tablets in the treatment of atherosclerosis cardiovascular disease by using network pharmacology and cell experimental validation. MethodThe components of Xinmaikang tablets were searched by BATMAN-TCM database and the active ingredients and potential targets were screened. The atherosclerosis related disease targets were searched in GeneCards and online mendelian inheritance in man（OMIM） disease databases. The therapeutic targets were obtained by mapping the intersection of the tablets and disease targets. Therapeutic targets were uploaded to STRING database to construct protein-protein interaction（PPI） network. Cytoscape software was used to create a "drug-active component-therapeutic target" network map， and a network topology algorithm was used to screen key action targets. David software was used for gene ontology（GO） and Kyoto encyclopedia of genes and genomes（KEGG） function enrichment analysis. The key targets of drug therapy were validated by in vitro cell assay. ResultA total of 19 active ingredients， 132 potential targets and 4703 atherosclerotic disease-related target genes of Xinmaikang tablets were retrieved and screened， and 84 intersection targets were obtained. 3 key therapeutic targets of Xinmaikang tablets in the treatment of atherosclerotic diseases were screened， including Calmodulin 1（CALM1）， voltage-dependent L-type calcium channel subunit alpha-1C（CACNA1C） and Phosphatidylinositol 4，5-bisphosphate 3-kinase catalytic subunit alpha isoform（PIK3CA）. A total of 313 biological processes， 89 molecular functions and 53 cell components were obtained by GO enrichment. A total of 40 pathways were obtained from KEGG functional enrichment， including purine metabolism， renin secretion， CGMP/PKG signaling pathway and so on. In vitro cell experiment results verified that Xinmaikang tablets can up-regulate the expression of CALM1 and CACNA1C， down-regulate the expression of PIK3CA， so as to inhibit the activity of inflammatory response， and play a therapeutic role in atherosclerotic diseases. ConclusionXinmaikang tablets may treat atherosclerosis cardiovascular disease through betulin， methyleugenol and other compounds， through purine metabolism， renin secretion， cGMP/PKG signaling pathway and other pathways， which acts on CALM1， CACNA1C， PIK3CA and other targets.
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ObjectiveTo predict the therapeutic target genes and related signaling pathways of Qinghuangsan （QHP） in the treatment of acute myeloid leukemia （AML） by network pharmacology，molecular docking，and further clarify its mechanisms through in vitro cell experiment. MethodThe active components and targets of QHP were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform （TCMSP），traditional Chinese medicine integrated database （TCMID），TargetNet and SwissTargetPrediction databases，and AML-related target genes were obtained by GeneCards and online mendelian inheritance in man （OMIM） databases. After screening the common targets of QHP and AML，the protein-protein interaction （PPI） network of the common targets was constructed with STRING，followed by gene ontology （GO） term and Kyoto encyclopedia of genes and genomes （KEGG） pathway enrichment analysis based on RStudio software and clusterProfiler，Bioconductor packages. At the same time，Cytoscape software is used to construct the network of "disease-component-target" and "compound-target-pathway". Select the active ingredients of QHP for molecular docking with the top 8 targets in the "compound-target-pathway" network. In vitro cell experiment and Western blot were used to further verify the anti-AML effect of QHP. ResultThe prediction results show that there are 11 main active components of QHP，and 22 common targets of QHP and AML are collected. KEGG pathway analysis results show that phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） and mitogen-activated protein kinase （MAPK） signaling pathways may play a key role in the treatment of AML disease by QHP. "Compound-target-pathway" network analysis showed that the top 8 targets include Akt1，phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit alpha （PIK3CA），mitogen-activated protein kinase kinase 1 （MAP2K1），TP53，serine/threonine kinase （RAF1），B cell lymphoma（Bcl）-2，cysteine aspartic acid specific protease（Caspase）-9 and JUN. Molecular docking results showed that 3-indolyl-β-D-glucopyranoside was optimally docked with MAP2K1，isovitexin docked with PIK3CA，and indirubin docked with Bcl-2. Cell experiments show that 3-indolyl-β-D-glucopyranoside，isovitexin and indirubin can effectively inhibit the proliferation of AML cells，regulate the MAPK/PI3K signaling pathway，and inhibit the expression of Bcl-2 protein. ConclusionQHP can treat AML through "multi-component，multi-target，multi-pathway" synergistic treatment，and its mechanism of pharmacology may be related to the regulation of MAPK signaling pathway and PI3K/Akt signaling pathway.
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@#Neuromyelitis optica(NMO)is an inflammatory central nervous system(CNS)astrocytic disease with high incidence, neuro-ophthalmic intercross, and humoral immune-dominated in Asian population. It has attracted much attention due to its high pathogenicity, high risk of recurrence, and poor prognosis. It is difficult for patients with NMO-associated optic neuritis(NMO-ON)to benefit from routine treatment, and they are often left with different degrees of optic nerve atrophy. One limitation of the study of NMO-ON is the deficiency of the experimental model. Therefore, the progress and application of NMO and NMO-ON experimental model are reviewed in this paper, aiming to explore the pathological mechanism and possible treatment of NMO visual impairment.
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Objective:To explore the active components， targets， and signaling pathways responsible for Bushen Zhuyun prescription in treating the recurrent spontaneous abortion （RSA） based on network pharmacology and uncover its potential mechanism by molecular docking and in vitro cell experiments. Method:The active components of Bushen Zhuyun prescription were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， Traditional Chinese Medicine Integrated Database （TCMID） and the published articles， followed by the prediction of drug action targets based on such platforms as DrugBank and SwissTargetPrediction. GeneCards and Online Mendelian Inheritance in Man （OMIM） were searched to obtain the RSA targets， which were then intersected with the targets of Bushen Zhuyun Decoction. Following the plotting of Bushen Zhuyun prescription-compound-target-RSA network by Cytoscape 3.7.1， the protein-protein interaction （PPI） network was then constructed with STRING for screening the core network. The resulting common targets were then subjected to Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis using R software. Autodock Vina 1.1.2 was used for molecular docking. The activation of phosphatidylinositol 3-kinase/protein kinase B （PI3K/AKT） signaling pathway by Bushen Zhuyun prescription was verified in human umbilical vein endothelial cells （HUVEC） <italic>in vitro</italic>. Result:It was found that 49 potential active components of Bushen Zhuyun prescription might act on 133 RSA targets. GO enrichment analysis yielded 470 biological processes， with angiogenesis， vascular development， cellular proliferation， and oxidative activity mainly involved. KEGG enrichment analysis revealed 103 signaling pathways （<italic>P</italic><0.05）， and the PI3K/AKT signaling pathway， advanced glycation end product （AGE）/receptor for advanced glycation end product （RAGE） signaling pathway， and tumor necrosis factor （TNF） signaling pathway were the main ones. As indicated by molecular docking， the Vina scores of the main active component kaempferol with AKT1 and vascular endothelial growth factor A （VEGFA） were the lowest and similar. It was confirmed <italic>in vitro</italic> cell experiments that Bushen Zhuyun prescription activated the PI3K/AKT signaling pathway and up-regulated the expression of VEGFA and downstream AKT protein to promote angiogenesis. Conclusion:Bushen Zhuyun prescription promotes angiogenesis at the maternal-fetal interface by regulating angiogenesis and cellular proliferation， activating the PI3K/AKT pathway， and up-regulated the VEGFA expression， which is beneficial to the formation of placenta in early pregnancy and the maintenance of early pregnancy. This study has provided ideas for new drug development.
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In this study, the molecular mechanism of astragaloside Ⅳ(AS-Ⅳ) in the treatment of Parkinson's disease(PD) was explored based on network pharmacology, and the potential value of AS-Ⅳ in alleviating neuronal injury in PD by activating the PI3 K/AKT signaling pathway was verified through molecular docking and in vitro experiments. Such databases as SwissTargetPrediction, BTMAN-TAM, and GeneCards were used to predict the targets of AS-Ⅳ for the treatment of PD. The Search Tool for the Retrieval of Interacting Genes/Proteins(STRING) was employed to analyze protein-protein interaction(PPI) and construct a PPI network, and the Database for Annotation, Visualization and Integrated Discovery(DAVID) was used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Based on the results of GO enrichment analysis and KEGG pathway analysis, the PI3 K/AKT signaling pathway was selected for further molecular docking and in vitro experiments in this study. The in vitro cell model of PD was established by MPP~+. The cell viability was measured by MTT assay and effect of AS-Ⅳ on the expression of the PI3 K/AKT signaling pathway-related genes and proteins by real-time polymerase chain reaction(RT-PCR) and Western blot. Network pharmacology revealed totally 122 targets of AS-Ⅳ for the treatment of PD, and GO enrichment analysis yielded 504 GO terms, most of which were biological processes and molecular functions. Totally 20 related signaling pathways were screened out by KEGG pathway analysis, including neuroactive ligand-receptor interaction, PI3 K/AKT signaling pathway, GABAergic synapse, and calcium signaling pathway. Molecular docking demonstrated high affinity of AS-Ⅳ to serine/threonine-protein kinases(AKT1, AKT2), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma(PIK3 CG), and phosphoinositide-3-kinase, catalytic, alpha polypeptide(PIK3 CA) on the PI3 K/AKT signaling pathway. In vitro experiments showed that AS-Ⅳ could effectively inhibit the decrease of the viability of PC12 induced by MPP~+ and up-regulate the mRNA expression levels of AKT1 and PI3 K as well as the phosphorylation levels of AKT and PI3 K. As an active component of Astragali Radix, AS-Ⅳ acts on PD through multiple targets and pathways. Furthermore, it inhibits neuronal apoptosis and protects neurons by activating the PI3 K/AKT signaling pathway, thereby providing reliable theoretical and experimental supports for the treatment of PD with AS-Ⅳ.
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Animals , Rats , Drugs, Chinese Herbal/pharmacology , Molecular Docking Simulation , Network Pharmacology , PC12 Cells , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Saponins , Signal Transduction , TriterpenesABSTRACT
Objective:To observe the effects of Chaihu Jiedu decoction on human hepatic stellate LX-2 cells,and to explore the potential molecular mechanisms.Methods:The wistar rats were divided into the experimental group and the control group,respectively with Chaihu Jiedudecoction and saline lavage,then centrifugal and get the drug-containing serum and control serum.At the same time,trsuscitate cells.When we got the expected number of cells,we divided into the experimental group and the control group.The human hepatic stellate cells (LX-2) were with the drug serum for 24 h,36 h,48 h,72 h.Then the cell proliferation inhibition rate was measured by CCK-8,the apoptosis were detected by flow cytometry (Annexin V-FITC,PI staining method).Results:ChaihuJiedu decoction could inhibit proliferation of LX-2 cells 24 hours after dosing.The in hibition rate in 36 h,48 h,72 h were 0.37 ％,0.46 ％,0.44 ％ respectively.It could prevent LX-2 cells into proliferation and induce the apoptosis of LX-2 cells.The apoptosis rates in 48 h,72 h were (9.80±0.95)％,(36.40± 5.09)％ respectively,and there are difference of statistical significance(P＜0.05).Conclusion:Chaihu Jiedu decoction can inhibit the proliferation of LX-2 cells proliferation,and induce apoptosis,so as to interfere with course of the liver fibrosis.