The binding of kaempferol-3-O-rutinoside to vascular endothelial growth factor potentiates anti-inflammatory efficiencies in lipopolysaccharide-treated mouse macrophage RAW264.7 cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Taken together, our results suggested the pharmacological roles of kaempferol-3-O-rutinoside in VEGF-C-mediated anti-inflammation.

Kaempferol, a Major Flavonoid in Ginkgo Folium, Potentiates Angiogenic Functions in Cultured Endothelial Cells by Binding to Vascular Endothelial Growth Factor.

Kaempferol is a major flavonoid in Ginkgo Folium and other edible plants, which is being proposed here to have roles in angiogenesis. Angiogenesis is important in both physiological and pathological development. Here, kaempferol was shown to bind with vascular endothelial growth factor (VEGF), probably in the heparin binding domain of VEGF: this binding potentiated the angiogenic functions of VEGF in various culture models. Kaempferol potentiated the VEGF-induced cell motility in human umbilical vein endothelial cells (HUVECs), as well as the sub-intestinal vessel sprouting in zebrafish embryos and formation of microvascular in rat aortic ring. In cultured HUVECs, application of kaempferol strongly potentiated the VEGF-induced phosphorylations of VEGFR2, endothelial nitric oxide synthase (eNOS) and extracellular signal-regulated kinase (Erk) in time-dependent and concentration-dependent manners, and in parallel the VEGF-mediated expressions of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were significantly enhanced. In addition, the potentiation effect of kaempferol was revealed in VEGF-induced migration of skin cell and monocyte. Taken together, our results suggested the pharmacological roles of kaempferol in potentiating VEGF-mediated functions should be considered.

Isoorientin derived from Gentiana veitchiorum Hemsl. flowers inhibits melanogenesis by down-regulating MITF-induced tyrosinase expression.

BACKGROUND: Gentiana is a genus of flowering plants in Gentianaceae family, which comprises of 1,600 species. The roots of few species of Gentiana, also known as Long Dan Cao in Chinese, are traditionally used in herbal remedies for a wide variety of liver-associated diseases. The medicinal part of Gentiana is root; however, the trumpet-shaped flowers are seldom being used. PURPOSE: We investigated the anti-melanogenesis effect of water extract of Gentiana veitchiorum Hemsl. flowers, and isoorientin was identified to be the active compound. STUDY DESIGN: We tested the anti-melanogenesis effects of extracts deriving from different parts of G. veitchiorum, followed by identification of active ingredients within the extracts. The mechanism of inhibitory effect on melanogenesis, triggered by isoorientin, was elucidated by in vitro analyses. METHODS: HPLC was applied to identify the components in water extracts from different parts of G. veitchiorum. The cytotoxicity of extracts and pure compounds in cultured B16F10 murine melanoma cells was determined by MTT and trypan blue assays. Melanin assay, tyrosinase assay, RT-PCR, luciferase assay and western blot were used to analyze the effect of isoorientin in melanin content, tyrosinase activity, as well as the expressions of those related genes and proteins. RESULTS: We identified an inhibitory effect on melanogenesis from water extract of G. veitchiorum flowers in B16F10 cells. Isoorientin, a major flavone in the extract, was identified to be an active ingredient causing reduction in melanin content in a dose-dependent manner. Such reduction was suggested to be a result of suppressed expression of tyrosinase (TYR), tyrosinase related protein-1 (TRP1) and DOPA-chrome tautomerase (DCT). Isoorientin also suppressed the expression of microphthalmia- associated transcription factor (MITF) through the phosphorylation of cAMP response element-binding protein (CREB). CONCLUSION: These findings indicate that isoorientin derived from G. veitchiorum flowers may be a potential skin lightening agent for the treatment of skin pigmentary disorders.

Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Polydatin, also called piceid, is a stilbenoid glucoside of a resveratrol derivative. It derives mainly from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. Although the role of P. cuspidatum root in angiogenesis has been reported, the active chemical or chemicals responsible for such function is not known. Here, polydatin was proposed to bind VEGF, which therefore altered the functions of VEGF in angiogenesis. Several lines of evidence supported the pharmaceutical effects of polydatin in VEGF-induced angiogenesis. In human umbilical vein endothelial cells, polydatin inhibited VEGF-stimulated cell proliferation, cell migration, and tube formation. Moreover, polydatin showed suppressive effects on the subintestinal vessel formation in zebrafish embryos. In signaling cascades, polydatin application attenuated VEGF-induced phosphorylations of VEGF receptor 2 and JNK. Moreover, the VEGF-induced phosphorylations of Akt, eNOS, and Erk were significantly decreased in the presence of polydatin. In parallel, the formation of reactive oxygen species, triggered by VEGF, was markedly decreased under polydatin application. Thus, our results supported the angiogenic roles of polydatin, as well as its signaling mechanism in blocking VEGF-mediated responses. The current study provides support for the possible development of polydatin as a potential therapeutic agent for treatment and prevention of angiogenesis-related diseases.-Hu, W.-H., Wang, H.-Y., Kong, X.-P., Xiong, Q.-P., Poon, K. K.-M., Xu, L., Duan, R., Chan, G. K.-L., Dong, T. T.-X., Tsim, K. W.-K. Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Binding of Resveratrol to Vascular Endothelial Growth Factor Suppresses Angiogenesis by Inhibiting the Receptor Signaling.

Resveratrol is a polyphenol commonly found in plants and food health products, such as grape and red wine, and was identified for its binding to vascular endothelial growth factor (VEGF) by using HerboChips screening. The binding, therefore, resulted in alterations of VEGF binding to its receptor and revealed the roles of VEGF in angiogenesis. Several lines of evidence gave support to the inhibitory activities of resveratrol in VEGF-triggered angiogenesis. In human umbilical vein endothelial cells (HUVECs), compared with a VEGF-induced group, resveratrol, at a high concentration, suppressed VEGF-mediated endothelial cell proliferation, cell migration, cell invasion, and tube formation by 80 ± 9.01%, 140 ± 3.78%, 110 ± 7.51%, and 120 ± 10.26%, respectively. Moreover, resveratrol inhibited the subintestinal vessel formation in zebrafish embryo. In signaling cascades, application of resveratrol in HUVECs reduced the VEGF-triggered VEGF receptor 2 phosphorylation and c-Jun N-terminal kinase phosphorylation. Moreover, the VEGF-mediated phosphorylations of endothelial nitric oxide synthase, protein kinase B, and extracellular signal-regulated kinase were obviously decreased by (3 ± 0.37)-, (2 ± 0.27)- and (6 ± 0.23)-fold, respectively, in the presence of resveratrol at high concentration. Parallelly, the VEGF-induced reactive oxygen species formation was significantly decreased by 50 ± 7.88% to 120 ± 14.82% under resveratrol treatment. Thus, our results provided support to the antiangiogenic roles of resveratrol, as well as its related signaling mechanisms, in attenuating the VEGF-mediated responses. The present results supported possible development of resveratrol, which should be considered as a therapeutic agent in terms of prevention and clinical treatment of diseases related to angiogenesis.

The extract of Polygoni Cuspidati Rhizoma et Radix suppresses the vascular endothelial growth factor-induced angiogenesis.

BACKGROUND: Polygoni Cuspidati Rhizoma et Radix (PCRR; the root and rhizome of Polygonum cuspidatum Sieb. et Zucc) is a traditional Chinese medicine for the treatment of inflammation, hyperlipemia, favus, jaundice and scald. HYPOTHESIS/PURPOSE: The extract of PCRR inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis. The hypothesis is supported by analysis of PCRR extract and investigation of pharmacological role and signaling mechanism of PCRR extract in regulating angiogenic responses. STUDY DESIGN: The PCRR ethanolic extract was examined for its inhibitory effects on angiogenesis based on VEGF-treated human umbilical vein endothelial cells and in zebrafish model METHODS: The effects and signaling mechanism of a standardized ethanolic extract of PCRR were tested on cell proliferation, migration and tube formation in VEGF-treated human umbilical vein endothelial cells, and which was further validated in zebrafish embryo model. RESULTS: The treatment of PCRR extract in cultured endothelial cells inhibited VEGF-induced cell proliferation, cell migration and tube formation in a dose-dependent manner and also suppressed the formation of sub-intestinal vessels in zebrafish embryos. Moreover, the applied PCRR extract suppressed VEGF-induced phosphorylations of VEGF receptor 2 (VEGFR2) and JNK. Thus, the site of effect triggered by PCRR was proposed to be mediated by VEGFR2. To further support this notion, the phosphorylations of Erk, Akt and eNOS, induced by VEGF, were markedly reduced under the challenge of PCRR extract: the reductions were subsequently further decreased in the present of inhibitors of Erk, Akt and eNOS. In parallel, the formation of ROS induced by VEGF in cultured endothelial cells was markedly reduced in the present of PCRR extract. CONCLUSION: Collectively, our studies demonstrated the pharmacological role and signaling mechanism of PCRR in regulation of angiogenic responses, which supported further evaluation and development of PCRR as a potential therapeutic agent for the treatment and prevention of diseases related with angiogenesis.

Indication of nerve growth factor binding components from herbal extracts by HerboChip: a platform for drug screening on a chip.

BACKGROUND: HerboChip is an array of different fractions deriving from herbal extracts. This study aimed to identify effective components from Chinese medicine (CM) that interact with nerve growth factor (NGF) as a target using HerboChip. METHODS: Fifty types of CM that are traditionally used as remedies for emotion imbalance were selected and extracted with 50 % ethanol. Biotinylated-NGF was hybridized with over 300 chips coated with different HPLC-separated fractions from CM extracts and straptavidin-Cy5 was used to identify the NGF-bound fractions. RESULTS: Over 300 chips were screened within a week, and 17 positive hits were identified. The interaction of the identified herbal extracts with NGF was confirmed in cultured PC12 cells. Co-application of NGF and herbal extract interfered with NGF-induced expression of neurofilaments, including NF68 and NF200 in cell cultures. Western blot analysis comparing the intensity of phosphorylated cAMP response element-binding protein (CREB) over total CREB showed NGF-induced CREB phosphorylation was modulated by the identified herbal extracts. Five CM herbs showed activating activities on the NGF response and nine CM herbs showed inhibiting activities. CONCLUSION: The current result supported the applicability of HerboChip for screening NGF binding components from herbal extracts.