kaempferol-3-o-rutinoside and Inflammation

Vascular Endothelial Growth Factors (VEGFs) are a group of growth factor in regulating development and maintenance of blood capillary. The VEGF family members include VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. VEGF receptor activation leads to multiple complex signaling pathways, particularly in inducing angiogenesis. Besides, VEGF is produced by macrophages and T cells, which is playing roles in inflammation. In macrophages, VEGF receptor-3 (VEGFR-3) and its ligand VEGF-C are known to attenuate the release of pro-inflammatory cytokines.. Immunoprecipitation and molecular docking assays showed the binding interaction of kaempferol-3-O-rutinoside and VEGF-C. Western blotting and qRT-PCR methods were applied to explore the potentiating effect of kaempferol-3-O-rutinoside in VEGF-C-mediated expressions of proteins and genes in endothelial cells and LPS-induced macrophages. Enzyme-linked immunosorbent assay (ELISA) was employed to reveal the release of proinflammatory cytokines in LPS-induced macrophages. Immunofluorescence assay was performed to determine the effect of kaempferol-3-O-rutinoside in regulating nuclear translocation of NF-κB p65 subunit in the VEGF-C-treated cultures. In addition, Transwell® motility assay was applied to detect the ability of cell migration after drug treatment in LPS-induced macrophages.. We identified kaempferol-3-O-rutinoside, a flavonoid commonly found in vegetable and fruit, was able to act on cultured macrophages in inhibiting inflammatory response, and the inhibition was mediated by its specific binding to VEGF-C. The kaempferol-3-O-rutinoside-bound VEGF-C showed high potency to trigger the receptor activation. In LPS-treated cultured macrophages, applied kaempferol-3-O-rutinoside potentiated inhibitory effects of exogenous applied VEGF-C on the secretions of pro-inflammatory cytokines, i.e. IL-6 and TNF-α, as well as expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). This inhibition was in parallel to transcription and translocation of NF-κB. Moreover, the binding of kaempferol-3-O-rutinoside with VEGF-C suppressed the LPS-induced migration of macrophage.. Taken together, our results suggested the pharmacological roles of kaempferol-3-O-rutinoside in VEGF-C-mediated anti-inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclooxygenase 2; Cytokines; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Kaempferols; Lipopolysaccharides; Macrophages; Mice; Molecular Docking Simulation; NF-kappa B; Nitric Oxide Synthase Type II; RAW 264.7 Cells; Vascular Endothelial Growth Factor C

Kaempferol‑3‑O‑β‑rutinoside is one of the compounds isolated from tartary buckwheat (Fagopyrum tatricum), and its biological effects have not been studied yet. The present study examined the anti‑inflammatory effects of kaempferol‑3‑O‑β‑rutinoside and explore its regulatory mechanisms in lipopolysaccharide (LPS)‑induced macrophage RAW264.7 cells. Kaempferol‑3‑O‑β‑rutinoside exhibited no cytotoxic effect in RAW 264.7 macrophage and 293 cell lines up to 300 µM. As the concentration of kaempferol‑3‑O‑β‑rutinoside was increased, the activity of nitric oxide was inhibited in LPS‑stimulated RAW264.7 cells. In addition, kaempferol‑3‑O‑β‑rutinoside treatment downregulated the expression of inflammation‑related cytokines tumor necrosis factor‑α and interleukin‑6 in LPS‑stimulated RAW264.7 cells. Furthermore, kaempferol‑3‑O‑β‑rutinoside treatment suppressed inflammatory‑mediated factors, such as inducible nitric oxide synthase and cyclooxyganse‑2. These inflammation‑related proteins are known to be regulated by NF‑κB and mitogen‑activated protein kinase (MAPK) signaling, therefore the effect of kaempferol‑3‑O‑β‑rutinoside on these pathways was investigated. The results demonstrated that kaempferol‑3‑O‑β‑rutinoside decreased the expression of inhibitor of κB (IκB) protein and IκB kinases; as a result, the nuclear translocation and expression of NF‑κB was inhibited in LPS‑stimulated RAW264.7 cells. Furthermore, kaempferol‑3‑O‑β‑rutinoside inhibited the phosphorylation of p38, extracellular signal‑regulated kinase and stress‑activated protein kinase in LPS‑stimulated RAW264.7 cells. Thus, the present data demonstrated that kaempferol‑3‑O‑β‑rutinoside suppressed inflammation‑related gene expression through the NF‑κB and MAPK pathways, and suggested that it may be a useful reagent in pharmacological research.

Topics: Animals; Cell Proliferation; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; Kaempferols; Lipopolysaccharides; Macrophages; MAP Kinase Signaling System; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorylation; RAW 264.7 Cells

Neuroinflammation is a key contributor to neuronal damage in neurodegenerative diseases. In our previous work on natural effective neuroinflammatory inhibitors, Alhagi sparsifolia Shap. (Leguminosae), a folk medicine widely distributed in Xinjiang, attracted our attention because of its significant anti-neuroinflammatory effect. Therefore, further investigation of the bioactive material basis was carried out. As a result, 33 major components were characterized and identified by chromatographic and spectral methods, respectively. Furthermore, the anti-neuroinflammatory effects of the extract and purified constituents were evaluated in LPS-induced N9 cells in vitro. The results displayed that compounds 1, 2, 3, 5, 6, 8, 11, 15, 16, 17, 22, 23, 25, 26, 28, 30, 33 could exhibit significant inhibitory activities without obvious cytotoxicities at their effective concentrations. Especially, isorhamnetin (1) (IC

Topics: Cell Line; Fabaceae; Humans; Inflammation; Lipopolysaccharides; Microglia; Neuroprotective Agents; Plant Extracts

Ischemic brain injury is associated with neuroinflammatory response, which essentially involves glial activation and neutrophil infiltration. Transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) contribute to ischemic neuroinflammatory processes and secondary brain injury by releasing proinflammatory mediators. Kaempferol-3-O-rutinoside (KRS) and kaempferol-3-O- glucoside (KGS) are primary flavonoids found in Carthamus tinctorius L. Recent studies demonstrated that KRS protected against ischemic brain injury. However, little is known about the underlying mechanisms. Flavonoids have been reported to have antiinflammatory properties. Herein, we explored the effects of KRS and KGS in a transient focal stroke model.. Rats were subjected to middle cerebral artery occlusion for 2 hours followed by 22 h reperfusion. An equimolar dose of KRS or KGS was administered i.v. at the beginning of reperfusion. The results showed that KRS or KGS significantly attenuated the neurological deficits, brain infarct volume, and neuron and axon injury, reflected by the upregulation of neuronal nuclear antigen-positive neurons and downregulation of amyloid precursor protein immunoreactivity in the ipsilateral ischemic hemisphere. Moreover, KRS and KGS inhibited the expression of OX-42, glial fibrillary acidic protein, phosphorylated STAT3 and NF-κB p65, and the nuclear content of NF-κB p65. Subsequently, these flavonoids inhibited the expression of tumor necrosis factor α, interleukin 1β, intercellular adhesion molecule 1, matrix metallopeptidase 9, inducible nitric oxide synthase, and myeloperoxidase.. Our findings suggest that postischemic treatment with KRS or KGS prevents ischemic brain injury and neuroinflammation by inhibition of STAT3 and NF-κB activation and has the therapeutic potential for the neuroinflammation-related diseases, such as ischemic stroke.

Topics: Animals; Astrocytes; Axons; Brain Injuries; Cerebral Infarction; Infarction, Middle Cerebral Artery; Inflammation; Inflammation Mediators; Kaempferols; Male; Microglia; Monosaccharides; Neuroprotective Agents; NF-kappa B; Rats; Rats, Sprague-Dawley; STAT3 Transcription Factor; Stroke

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature