Anti-inflammatory effects of ivy leaves dry extract: influence on transcriptional activity of NFκB.

EA 575® is an ivy leaves dry extract (DER 5-7.5:1, 30% ethanol) used against diseases of the lower respiratory tract associated with productive cough. EA 575® improves symptoms associated with chronic inflammatory bronchial conditions. Compared to its bronchospasmolytic and secretolytic properties, the anti-inflammatory effects of EA 575® are mostly untried. Therefore, we addressed the question of whether the anti-inflammatory effect of EA 575® is due to an impact on the NFκB pathway. NFκB nuclear translocation was visualized by immunofluorescence in J774.2 as well as HEK293 cells. In the latter, a luciferase-based reporter was used to monitor NFκB transcriptional activity. Phosphorylation of RelA and its inhibitor IκB was measured by Western blot analysis. Additionally, changes in the stability of NFκB:IκB complex were shown by protein fragment complementation. Decreased transcriptional activity of NFκB under treatment with EA 575® was also shown for a human monocytic as well as a human lung epithelial cell line. EA 575® is able to inhibit NFκB transcriptional activity by partially inhibiting its translocation to the nucleus after stimulation with TNFα. Furthermore, phosphorylation of IκBα is reduced while phosphorylation of RelA is enhanced after pre-incubation with EA 575®, leading to an enhanced stability of NFκB:IκBα complex. EA 575® has an regulatory impact on the NFκB pathway, possibly by switching specificity of IKK from IκBα to RelA, resulting in enhanced stability of NFκB:IκBα complex and reduced RelA translocation into the nucleus.

Ivy leaves dry extract EA 575® mediates biased ß2-adrenergic receptor signaling.

BACKGROUND: ß2-adrenergic receptor (ß2-AR) stimulation activates the G protein/cAMP pathway, which is opposed by the GRK2/ß-arrestin 2 pathway. The latter is undesirable in the treatment of respiratory diseases. HYPOTHESIS/PURPOSE: EA 575® is capable of mediating a biased ß2-adrenergic signaling pathway. METHODS: The impact of the ivy leaves dry extract EA 575® on ß2-adrenergic signaling was tested in a dynamic mass redistribution assay in HEK wild-type and in HEK ß-arrestin knock-out cells. cAMP formation and recruitment of ß-arrestin 2 were investigated using GloSensor™ and PathHunter® assays, respectively. NFκB transcriptional activity was determined in both HEK wild-type as well as HEK ß-arrestin knock-out cells. RESULTS: EA 575® inhibits the recruitment of ß-arrestin 2 and thereby enhances G protein/cAMP signaling under ß2-stimulating conditions, as evidenced by a corresponding increase in cAMP formation. While ß2-AR-mediated inhibition of NFκB transcriptional activity is ß-arrestin-dependent, EA 575® leads to significant inhibition of NFκB transcriptional activity in ß-arrestin knock-out cells and thus via a ß-arrestin-independent signaling pathway. CONCLUSION: EA 575® is the first active phytopharmaceutical ingredient for which biased ß2-adrenergic activation has been described. This shift towards G protein/cAMP signaling provides the molecular basis for the clinically proven efficacy of EA 575® in the treatment of lower respiratory tract diseases. In this light, EA 575® could potentially reduce ß-arrestin-mediated adverse effects in new combinatorial therapeutic approaches.

A comparative study on the influence of an ivy preparation and an ivy/thyme combination on the ß2-adrenergic signal transduction.

The ß2-adrenergic receptor (ß2AR) is relevant for surfactant formation in alveolar type 2 cells and reduction of intracellular calcium concentration in bronchial muscle cells and thus for secretolytic and bronchospasmolytic effects. Herbal medicinal products that affect the ß2AR system are used to treat common cold and bronchitis accompanied with mucus covered and narrowed airways. The present work compares the influence of an ivy preparation and an ivy/thyme combination on the ß2-adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of ß2AR we have used single molecule detection by fluorescence correlation spectroscopy and single particle tracking. For the determination of both the second messenger cAMP and the internalization of ß2AR we have generated luciferase based reporter cell lines, which produce a cAMP-dependent luciferase in the cytosol and express ß2AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the ß2AR binding, a significant increase of the cAMP level was observed only for the ivy preparation, which can be explained by the inhibited internalization of HiBiT-tagged ß2AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged ß2AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß2AR internalization either. Furthermore, SNAP-tagged ß2AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing ß2AR. A corresponding redistribution of these receptors was not observed after pre-treatment with both the ivy/thyme combination and the thyme preparation. Comparable to the ivy/thyme combination, no decrease in the intratrack transitioning probability ratio (p23/p32) for fast and slow diffusing ß2AR was found for the thyme preparation, which, however, significantly decreased for control cells and for pre-treatment with the ivy preparation under stimulating conditions. It can therefore be concluded that the thyme fluid extract fraction in the ivy/thyme combination may have in part a negative effect on the ß2-adrenergic signal transduction.

α-Hederin inhibits G protein-coupled receptor kinase 2-mediated phosphorylation of ß2-adrenergic receptors.

BACKGROUND: Recently is has been shown that α- and ß-hederin increase the ß2-adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of ß2-adrenergic receptors (ß2AR) under stimulating conditions. Internalization of ß2AR is initiated by phosphorylations of certain serines and threonines by cAMP dependent protein kinase A (PKA) and G protein-coupled receptor kinases (GRK). PURPOSE: To evaluate the effect of α-hederin on PKA and GRK2 mediated phosphorylation of GFP-tagged ß2AR. STUDY DESIGN: To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing ß2AR as GFP fusion protein and specific antibodies against PKA (Ser345/346) and GRK2 (Ser355/356) phosphorylation sites. RESULTS: There was no effect found on the PKA mediated phosphorylation (n = 14) but we could show that α-hederin (1 µM, 12 h) significantly inhibits GRK2 mediated phosphorylation at Ser355/356 by 11 ± 5% (n ≥ 29, p ≤ 0.01) under stimulating conditions compared to the positive control. In Förster resonance energy transfer (FRET) experiments using the isolated kinases in solution α-hederin did not show any influence neither to GRK2 nor to PKA. CONCLUSION: Taken together, these results indicate that α-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of ß2AR-GFP in HEK293 cells.