No abstract available.
Adiponectin ; Caspase 14 ; Humans ; Keratinocytes

To provide experimental basis and theoretical guidance for further research on the molecular mechanism of notoginsenoside R_1(NGR_1) in the treatment of depression, the present study analyzed the potential mechanism of NGR_1 in the treatment of depression through network pharmacology and verified it by molecular docking and animal experiments. PharmMapper, SwissTargetPrediction, and GeneCards were used to predict the related targets of both NGR_1 and depression to obtain the potential targets of NGR_1 in the treatment of depression. The database for annotation, visualization and integrated discovery(DAVID) was used for GO functional annotation and KEGG pathway enrichment analysis to screen the possible mechanisms of NGR_1 exerting antidepressant effect. Cytoscape 3.9.0 was adopted to construct a protein-protein interaction(PPI) network, and the topological analysis was performed to obtain the core targets. The binding activity of NGR_1 to core targets was tested by molecular docking. The depression model was prepared by injecting lipopolysaccharide(LPS) into the lateral ventricle in mice, and intervened with NGR_1. The antidepressant effect of NGR_1 was detected by behavioral tests and RT-qPCR. The results showed that by network pharmacology, 56 common targets of NGR_1 and depression were predicted, and GO enrichment analysis determined 13 related biological processes, mainly involving G protein-coupled receptor signaling pathway, positive regulation of transcription from RNA polymerase Ⅱ promoter, cytokine-mediated signaling pathway, gene expression, apoptosis, cell proliferation, and signal transduction. In addition, KEGG pathway enrichment analysis identified ten potential pathways, including neuroactive ligand-receptor interaction signaling pathway, lipid and atherosclerosis signaling pathway, cAMP signaling pathway, PI3 K-AKT signaling pathway, and lipid and atherosclerosis signaling pathway. PPI analysis revealed that the core targets included CASP3, VEGFA, IGF1, STAT3, MAPK1, PPARG, MTOR, MAPK14, NR3 C1 and AR, and molecular docking demonstrated that NGR_1 had desirable binding activity to these target proteins. In animal experiments, the results showed that NGR_1 improved the disease behavior of depressed mice, significantly inhibited the neuroinflammatory response(reducing the mRNA expression of Iba-1, TNF-α, IL-1β, and IL-6), and regulated the mRNA expression of lipid and atherosclerosis signaling pathway-related targets(CASP3, STAT3, MAPK1 and MAPK14). This indicated that the antidepressant mechanism of NGR_1 may be related to the regulation of lipid and atherosclerosis signaling pathway. In conclusion, network pharmacology was used to reveal the core targets and pathways of NGR_1, and some of them were verified in animal experiments, which provided the basis for in-depth exploration on the mechanism of NGR_1 in the treatment of depression.
Mice ; Animals ; Caspase 3 ; Animal Experimentation ; Mitogen-Activated Protein Kinase 14 ; Network Pharmacology ; Molecular Docking Simulation ; Antidepressive Agents/pharmacology* ; Lipopolysaccharides ; Atherosclerosis ; RNA, Messenger ; Drugs, Chinese Herbal/pharmacology*

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.
bcl-Associated Death Protein/*metabolism ; *Signal Transduction/drug effects ; Mitochondria/drug effects ; Mice ; Mastocytoma ; Hydrocarbons, Fluorinated/*pharmacology ; Genistein/pharmacology ; Enzyme Inhibitors/*pharmacology ; Cell Line, Tumor ; Caspase 6/antagonists & inhibitors/*metabolism ; Benzyl Compounds/*pharmacology ; Apoptosis/*drug effects ; Animals ; Amino Acid Chloromethyl Ketones/pharmacology ; 14-3-3 Proteins/*metabolism

BACKGROUNDAtopic dermatitis (AD) is characterized by defective skin barrier and imbalance in T helper 1/T helper 2 (Th1/Th2) cytokine expression. Filaggrin (FLG) is the key protein to maintaining skin barrier function. Recent studies indicated that Th1/Th2 cytokines influence FLG expression in keratinocytes. However, the role of Th1/Th2 cytokines on FLG processing is not substantially documented. Our aim was to investigate the impact of Th1/Th2 cytokines on FLG processing.

METHODSHaCaT cells and normal human keratinocytes were cultured in low and high calcium media and stimulated by either interleukin (IL)-4, 13 or interferon-γ (IFN-γ). FLG, its major processing proteases and key protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) were measured by both real-time quantitative polymerase chain reaction and Western blotting. Their expression was also evaluated in acute and chronic AD lesions by immunohistochemistry.

RESULTSIL-4/13 significantly reduced, while IFN-γ significantly up-regulated FLG expression. IL-4/13 significantly increased, whereas IFN-γ significantly decreased the expression of kallikreins 5 and 7, matriptase and channel-activating serine protease 1. On the contrary, IL-4/13 significantly decreased, while IFN-γ increased the expression of LEKTI and caspase-14. Similar trends were observed in AD lesions.

CONCLUSIONSOur results suggested that Th1/Th2 cytokines differentially regulated the expression of major FLG processing enzymes. The imbalance between Th1 and Th2 polarized immune response seems to extend to FLG homeostasis, through the network of FLG processing enzymes.

Caspase 14 ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Dermatitis, Atopic ; metabolism ; Humans ; Immunohistochemistry ; Interferon-gamma ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; metabolism ; Intermediate Filament Proteins ; metabolism ; Keratinocytes ; enzymology ; metabolism ; Proteinase Inhibitory Proteins, Secretory ; metabolism ; Serine Peptidase Inhibitor Kazal-Type 5 ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism