The Effect of Ivy Leaf Dry Extract EA 575® on the Signalling of Adenosine Receptor A2B
The Effect of Ivy Leaf Dry Extract EA 575® on the Signalling of Adenosine Receptor A2B
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DissertationPrüfungsdatum
16.05.2024Datum der Veröffentlichung
23.05.2024Erstgutachter
Häberlein, HannsZweitgutachter
Bendas, GerdMetadaten
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Inhalt
Ivy leaf dry extract EA 575® is used to alleviate the symptoms associated with chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing β2-adrenergic signal transduction. The present study investigated the effect of EA 575® on the signalling of adenosine receptor A2B (A2BAR), as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. In addition, this study approached to identify the compounds of EA 575® responsible for the observed effects. Moreover, a HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was generated, with the aim to conduct experiments on the receptor’s behaviour under the influence of EA 575®.
The influence of EA 575® on A2BAR signalling was assessed using various cellular assays targeting different events in the signalling cascade. Initially, the effect on the cellular reaction following A2BAR stimulation was investigated by measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via LumitTM Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified. Bioassay-guided fractionation of EA 575® was performed, combining HPLC methods with cAMP measurements and LC-MS/MS analyses, for the identification and isolation of bioactive compounds. CRISPR/Cas9-mediated homology-directed repair (HDR) was utilised for the expression of HiBiT-tagged A2BAR under endogenous promotion in HEK293 cells.
The results demonstrate the inhibitory effect of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occurred within a time frame of several hours of incubation, its mode of action may be described as indirect. The data presented here are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. Thus, a novel mechanism of action of EA 575® was revealed. Additionally, two bioactive sub-fractions of EA 575® were obtained that showed effects similar to the total extract, allowing to narrow down potential candidates responsible for the newly discovered effects of EA 575®. The HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was successfully generated using CRISPR/Cas9-mediated HDR. However, due to insufficient signals, it could not be utilised for the additional experiments on the receptor’s behaviour.
The findings of this study offer an explanation for initial positive clinical effects associated with EA 575® in adjuvant asthma therapy. Furthermore, they provide a rationale to conduct additional studies in animals or human subjects to explore further effects on chronic airway diseases such as asthma or COPD.
The influence of EA 575® on A2BAR signalling was assessed using various cellular assays targeting different events in the signalling cascade. Initially, the effect on the cellular reaction following A2BAR stimulation was investigated by measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via LumitTM Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified. Bioassay-guided fractionation of EA 575® was performed, combining HPLC methods with cAMP measurements and LC-MS/MS analyses, for the identification and isolation of bioactive compounds. CRISPR/Cas9-mediated homology-directed repair (HDR) was utilised for the expression of HiBiT-tagged A2BAR under endogenous promotion in HEK293 cells.
The results demonstrate the inhibitory effect of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occurred within a time frame of several hours of incubation, its mode of action may be described as indirect. The data presented here are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. Thus, a novel mechanism of action of EA 575® was revealed. Additionally, two bioactive sub-fractions of EA 575® were obtained that showed effects similar to the total extract, allowing to narrow down potential candidates responsible for the newly discovered effects of EA 575®. The HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was successfully generated using CRISPR/Cas9-mediated HDR. However, due to insufficient signals, it could not be utilised for the additional experiments on the receptor’s behaviour.
The findings of this study offer an explanation for initial positive clinical effects associated with EA 575® in adjuvant asthma therapy. Furthermore, they provide a rationale to conduct additional studies in animals or human subjects to explore further effects on chronic airway diseases such as asthma or COPD.
Schlagwörter
Efeu, EA 575, A2BAR, IL-6, HPLC, CRISPR/Cas9, Ivy
Klassifikation (DDC)
570 Biowissenschaften, BiologieZugehörige Publikation(en)
https://doi.org/10.3390/ijms241512373Meurer, Fabio Gian-Luca: The Effect of Ivy Leaf Dry Extract EA 575® on the Signalling of Adenosine Receptor A2B. - Bonn, 2024. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-76229
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-76229
@phdthesis{handle:20.500.11811/11565,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-76229,
author = {{Fabio Gian-Luca Meurer}},
title = {The Effect of Ivy Leaf Dry Extract EA 575® on the Signalling of Adenosine Receptor A2B},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = may,
note = {Ivy leaf dry extract EA 575® is used to alleviate the symptoms associated with chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing β2-adrenergic signal transduction. The present study investigated the effect of EA 575® on the signalling of adenosine receptor A2B (A2BAR), as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. In addition, this study approached to identify the compounds of EA 575® responsible for the observed effects. Moreover, a HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was generated, with the aim to conduct experiments on the receptor’s behaviour under the influence of EA 575®.
The influence of EA 575® on A2BAR signalling was assessed using various cellular assays targeting different events in the signalling cascade. Initially, the effect on the cellular reaction following A2BAR stimulation was investigated by measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via LumitTM Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified. Bioassay-guided fractionation of EA 575® was performed, combining HPLC methods with cAMP measurements and LC-MS/MS analyses, for the identification and isolation of bioactive compounds. CRISPR/Cas9-mediated homology-directed repair (HDR) was utilised for the expression of HiBiT-tagged A2BAR under endogenous promotion in HEK293 cells.
The results demonstrate the inhibitory effect of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occurred within a time frame of several hours of incubation, its mode of action may be described as indirect. The data presented here are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. Thus, a novel mechanism of action of EA 575® was revealed. Additionally, two bioactive sub-fractions of EA 575® were obtained that showed effects similar to the total extract, allowing to narrow down potential candidates responsible for the newly discovered effects of EA 575®. The HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was successfully generated using CRISPR/Cas9-mediated HDR. However, due to insufficient signals, it could not be utilised for the additional experiments on the receptor’s behaviour.
The findings of this study offer an explanation for initial positive clinical effects associated with EA 575® in adjuvant asthma therapy. Furthermore, they provide a rationale to conduct additional studies in animals or human subjects to explore further effects on chronic airway diseases such as asthma or COPD.},
url = {https://hdl.handle.net/20.500.11811/11565}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-76229,
author = {{Fabio Gian-Luca Meurer}},
title = {The Effect of Ivy Leaf Dry Extract EA 575® on the Signalling of Adenosine Receptor A2B},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = may,
note = {Ivy leaf dry extract EA 575® is used to alleviate the symptoms associated with chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing β2-adrenergic signal transduction. The present study investigated the effect of EA 575® on the signalling of adenosine receptor A2B (A2BAR), as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. In addition, this study approached to identify the compounds of EA 575® responsible for the observed effects. Moreover, a HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was generated, with the aim to conduct experiments on the receptor’s behaviour under the influence of EA 575®.
The influence of EA 575® on A2BAR signalling was assessed using various cellular assays targeting different events in the signalling cascade. Initially, the effect on the cellular reaction following A2BAR stimulation was investigated by measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via LumitTM Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified. Bioassay-guided fractionation of EA 575® was performed, combining HPLC methods with cAMP measurements and LC-MS/MS analyses, for the identification and isolation of bioactive compounds. CRISPR/Cas9-mediated homology-directed repair (HDR) was utilised for the expression of HiBiT-tagged A2BAR under endogenous promotion in HEK293 cells.
The results demonstrate the inhibitory effect of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occurred within a time frame of several hours of incubation, its mode of action may be described as indirect. The data presented here are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. Thus, a novel mechanism of action of EA 575® was revealed. Additionally, two bioactive sub-fractions of EA 575® were obtained that showed effects similar to the total extract, allowing to narrow down potential candidates responsible for the newly discovered effects of EA 575®. The HEK293 cell line expressing HiBiT-tagged A2BAR at an endogenous expression level was successfully generated using CRISPR/Cas9-mediated HDR. However, due to insufficient signals, it could not be utilised for the additional experiments on the receptor’s behaviour.
The findings of this study offer an explanation for initial positive clinical effects associated with EA 575® in adjuvant asthma therapy. Furthermore, they provide a rationale to conduct additional studies in animals or human subjects to explore further effects on chronic airway diseases such as asthma or COPD.},
url = {https://hdl.handle.net/20.500.11811/11565}
}
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