br Materials and methods br Results

Materials and methods
Results Our data indicated that 1,8-cineol leads to a noticeable but not significant (p = 0.07) 45% decreased phosphorylation of GSK-3α/β at Ser-9/21. Phosphorylation of GSK-3α/β at Tyr-279/216 was not affected (Fig. 1A/B). To ensure, that the decreased phosphorylation of GSK-3α/β at Ser-9/21 was not cause by a decreased GSK-3α/β expression, we adjusted phosphorylation levels also to GSK-3α/β. We could confirm previous data, 1,8-cineol leads to a 31% decreased phosphorylation of GSK-3α/β at Ser-9/21 when normalized to GSK-3α/β. Phosphorylation of GSK-3α/β at Tyr-279/216 was also not affected (Fig. 1C). Our immunohistochemical investigations demonstrated a decreased Ro 08-2750 of p-GSK-3 (Ser9/21) in response to 100 µM 1,8-cineol, which confirmed previous experiments (Fig. 2). In the next step we analyzed the expression level of active β-catenin, which is the central transcriptional activator protein of the Wnt signaling pathway. Active β-catenin protein almost disappeared in all analyzed tissue samples, so our data revealed a significant (p ≤ 0.05) decrease of active β-catenin in nasal polyps in response to 1,8-cineol treatment compared to medium controls (Fig. 3A/B). In this study, we additionally analyzed the expression- and Ser473 phosphorylation levels of Akt, as well as the expression of SGK1, SGK2 and SGK3, the negative regulators of GSK3-β activity, in response to 100 µM 1,8-cineol. The expression of Akt, SGK1, SGK2, SGK3 and phosphorylation levels of Akt at Ser473 were not significantly affected in all analyzed nasal polyp samples (Figs. 4A/B, 5).
Discussion Chronic rhinosinusitis with nasal polyps is a significant health problem and a multifunctional disease with an unclear proliferative benign nature. The current standard treatment recommended by the International and European consensus papers is topical corticosteroids. The response of nasal polyps to steroids, however, even if applied as long-term therapy with intermittent short courses of systemic glucocorticoids, frequently remains unsatisfactory and thus surgery often becomes necessary (Fokkens et al., 2012). Glucocorticosteroids have anti-inflammatory effects through protein-protein interactions, but longer applications increased the side effects. Herbal medications could reduce these side effects. In nature, essential oils are used in man because of their secretolytic properties, but irritation of the airway following inhalation or ingestion has limited their clinical use. 1,8-cineol, the major constituent of eucalyptus oil, was used for the treatment of acute and chronic bronchitis, sinusitis, and respiratory infections and is well tolerated. 1,8-cineol, a monoterpene, is an isoprenoid with anti-inflammatory effects and related to human isoprenoids like glucocorticosteroid (Juergens et al., 2003), but the mechanisms of action of 1,8-cineol are not fully understood. In this study, we have a closer look at the impact of 1,8-cineol on the regulation of the Wnt/β-catenin signaling pathway, especially its central regulator protein GSK-3. GSK-3 is a regulator of the innate and adaptive immune response and controls more than 20 transcription factors, which are important for immune function (Beurel et al., 2010). This multitasking enzyme, which is involved in numerous cellular signaling cascades, is located downstream of several major signaling pathways including the phosphatidylinositol 3′ kinase pathway, the Wnt pathway, Hedgehog signaling and Notch (Kaidanovich-Beilin and Woodgett, 2011). In previous studies of our group, we could show a significantly higher phosphorylation rate of GSK-3α/β at Ser9/21 in nasal polyps compared to the inferior turbinate of patients with CRSwNP and thus an inactivation of GSK-3 in native nasal polyp tissues (Linke et al., 2013). A deregulated GSK-3 activity have been demonstrated by many studies and was implicated in the pathogenesis and progression of different diseases, like neurodegenerative disorders, immunological diseases and chronic inflammation, but also in cancer. GSK-3 has been put forward to be an attractive target for a broad spectrum of disease (Beurel et al., 2015, Beurel et al., 2010, Domoto et al., 2016, Takahashi-Yanaga, 2013).