Mechanisms of action that contribute to the efficacy of ivy leaves dry extract EA 575^®

The ivy leaves dry extract EA 575® is an approved cough remedy distributed in more than 100 countries worldwide. The modes of action for its bronchospasmolytic, secretolytic, and anti-inflammatory effects remain poorly understood. Therefore, this work focuses on a contribution to these mechanisms. Within this work it is shown that the former investigated inhibition of β2-adrenergic receptor internalization arrives from an indirect inhibition of receptor phosphorylation by GRK2. Also the recruitment of β-arrestin2 to the β2-adrenergic receptor (β2AR) is shown to be diminished after EA 575® pre-treatment under stimulating conditions. With respect to anti-inflammatory effects, EA 575® reduces IL-6 secretion from murine macrophages after LPS stimulation. This effect is explained by a reduced NFκB transcriptional activity measured by a reporter gene assay based on luciferase activity in HEK and THP-1 cells. As assessed by immunostaining, the translocation of NFκB into the nucleus under stimulating conditions is also impaired, which is explained by a higher amount of bound NFκB to its inhibitor IκBα detected by a protein fragment complementation assay. This result is underlined with a lowered phosphorylation of IκBα and a heightened phosphorylation of NFκB subunit RelA at Ser536 after EA 575® pre-treatment and TNFα stimulation. Finally, it is demonstrated in HEK Nanoluc-PEST cells that NFκB transcriptional activity is diminished by β2-adrenergic stimulation and synergistically inhibited by EA 575® pre-incubation. Interestingly, this effect is shown to be β-arrestin dependent. Whether or not this crosstalk contributes to the efficacy of EA 575® requires further investigation