Inhibitory effects of casticin on migration of eosinophil and expression of chemokines and adhesion molecules in A549 lung epithelial cells via NF-κB inactivation

Abstract

Ethnopharmacological relevance

The fruits of Vitex rotundifolia L. have long been used for the treatment of inflammation of the respiratory tract in East Asia.

Aim

To determine if casticin, one of the constituents of Vitex rotundifolia L., has anti-allergic and anti-inflammatory effects in asthma.

Materials and methods

The in vitro anti-inflammatory activity of casticin was studied in A549 human type II-like epithelial lung cells using an eotaxin inhibition assay. Additionally, its effects on eotaxin, regulated on activation normal T cell expressed and secreted (RANTES), vascular cell adhesion molecule (VCAM)-1, and inter-cellular adhesion molecule (ICAM)-1 expression were investigated by real time-polymerase chain reaction (real time-PCR). The inhibition of nuclear factor κB (NF-κB) activity in the presence of casticin was determined by analyzing confocal microscopy images of fluorescence immunocytochemical analysis while the suppression of inhibitory κB (IκB)-α phosphorylation was studied using Western blot analysis. Finally, the inhibitory effect of casticin on eosinophil migration toward prestimulated A549 cell media was measured using the human eosinophilic leukemia cell line.

Results and discussion

Casticin significantly suppressed eotaxin production in cytokine activated A549 lung epithelial cells. Casticin also suppressed the mRNA expression levels of eotaxin, RANTES, VCAM-1, and ICAM-1, which subsequently contributed to the inhibition of eosinophil migration. Furthermore, casticin inhibited IκB-α phosphorylation and nuclear translocation of p65 in A549 cells.

Conclusion

Casiticin inhibited the eosinophil migration and activity of chemokines and adhesion molecules involved in the inflammatory process of asthma by suppressing the NF-κB pathway. These results suggest that casticin has the potential for use in the treatment of allergic asthma.

Introduction

The prevalence of asthma is increasing globally and is responsible for morbidity in all age groups, especially young children (Braman, 2006). Allergic inflammation is a distinctive feature of asthma that is characterized by infiltration and accumulation of lymphocytes, mast cells, and particularly, eosinophils into lung tissue (Cockcroft and Davis, 2006).

Under baseline conditions, eosinophils are produced and differentiate in bone marrow, after which they leave and migrate to the gastrointestinal tract, where they reside within the lamina propria (Rothenberg, 1998). However, under asthmatic conditions, eosinophils migrate from peripheral blood to respiratory endothelial cells, causing bronchial responsiveness and airway remodeling (Kay, 1991). Thus, the severity of asthma symptoms is closely related to the degree of eosinophil accumulation in the airway (Djukanovi et al., 1992).

The recruitment and infiltration of eosinophils into the lung are controlled by chemokines and adhesion molecules. Eotaxin is a major eosinophil-specific chemoattractant produced in the respiratory epithelium during allergic inflammation (Gangur and Oppenheim, 2000). Regulated on activation normal T cell expressed and secreted (RANTES) also plays a key role in facilitating migration and adhesion of eosinophils to the site of inflammation (Elsner et al., 2004). Moreover, adherence of eosinophils to vascular endothelial cells via adhesion molecule is considered to be essential in eosinophilic allergic inflammation. Vascular cell adhesion molecule (VCAM)-1 is expressed on activated endothelial cells and contributes to the firm adhesion of eosinophils to vascular endothelial cells (Foster, 1996). Inter-cellular adhesion molecule (ICAM)-1 is an endothelial transmembrane protein that facilitates endothelial transmigration of eosinophils into different pathologic situations (Zhu et al., 2000).

Nuclear factor-κB (NF-κB) is a well-known transcriptional factor that regulates airway inflammation and eosinophil activation. In unstimulated conditions, NF-κB heterodimers consisting of a p50 or p52 subunit and p65 dimers are sequestered in the cytoplasm by inhibitory κB (IκB). Since IκB masks the nuclear localization signals of NF-κB proteins, NF-κB remains sequestered in an inactive state in the cytoplasm. However, IκBα is phosphorylated under inflammatory conditions, which results in the release of NF-κB dimers from the cytoplasmic NF-κB-IκB complex and allows NF-κB to translocate to the nucleus. NF-κB then binds to κB enhancer elements of target genes, inducing those responsible for the transcription of a wide variety of genes involved in the inflammatory process (Ghosh et al., 1998).

Vitex rotundifolia L. (Verbenaceae) grows in the Mediterranean region, Central Asia, and along the seacoast from south to north of China. The fruits of Vitex rotundifolia L. have been used for medicinal purposes, particularly treatment of inflammation of the respiratory system (CK et al., 1996). Recently, scientific evidences of its anti-nociceptive, anti-inflammatory characteristics have supported its potential for the treatment of inflammatory disease (Shin et al., 2001, Hu et al., 2007, Sohn et al., 2009). Casticin, one of chemical components of Vitex rotundifolia L., has been reported to have anti-oxidant, anti-inflammatory, and immunomodulatory characteristics (You et al., 1998, Alam et al., 2002, Remberg et al., 2004, Lin et al., 2007, Choudhary et al., 2009, Mesaik et al., 2009). However, almost no information is available regarding the underlying mechanism of anti-allergic and anti-inflammatory effects of casticin in asthma. Therefore, we conducted this study to investigate the inhibitory effects of casticin on eosinophil migration and chemokines and adhesion molecules involved in the inflammatory responses of asthma. We then evaluated the suppressive effect of casticin on the activation of NF-κB to reveal its mode of mechanism.