Figure 6. Effects of artesunate on EGF stimulation of normal human bronchial epithelial cells. (A) Epithelial cells were stimulated with 100 ng/ml EGF in the presence and absence of 10 mM artesunate for 5, 15 and 30 minutes before total proteins were extracted for subsequent immunoblotting analysis. b-actin was used as an internal control. (B) DNA-binding activity of p65 NF-kB in nuclear extracts of epithelial cells stimulated with EGF for 30 minutes in the presence and absence of 10 mM artesunate was determined using a TransAMTM p65 transcription factor ELISA kit. (C) Epithelial cells were stimulated with 100 ng/ml EGF in the presence and absence of 10 mM artesunate for 12 hours before total mRNA was extracted using TriZol reagent. PCR products were separated in a 2% agarose gel and visualized under UV light. b-actin was used as an internal control. Representative gels from 4 separate experiments with similar pattern of results. Values shown are the mean 6 SEM of three separate experiments. *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g006 Artesunate Attenuates Asthma Figure 7. Effects of artesunate on house dust mite extracts- induced pulmonary inflammatory cell recruitment. Inflammatory cell counts in BAL fluid obtained from mice 72 hours after the last house dust mitechallenge (n = 6 mice) markedly increased as compared to saline control (n = 4 mice). Artesunate (30 mg/kg, n = 6 mice) significantly suppressed house dust mite-induced increases in BAL fluid total cell and eosinophil counts as compared with vehicle control (DMSO, n = 5). Differential cell counts were performed on a minimum of 500 cells to identify (Eos), macrophage (Mac), neutrophil (NEU), and lymphocyte (Lym). *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g007 Th2 cytokines play an essential role in the pathogenesis of the allergic airway inflammation [1,2]. IL-4, IL-5 and IL-13 can be produced by various lung resident cells such as bronchial epithelial cells, mast cells and alveolar macrophages as well as infiltrated inflammatory cells such as lymphocytes and eosinophils. Our present results show that artesunate significantly reduced the level of IL-4, IL-5, IL-13 and eotaxin in BAL fluids from OVA- challenged mice. They are in line with studies using mice deficient in p110d or p110c PI3K showing a major drop in Th2 cytokines and chemokines upon allergen sensitization and challenge [21,22]. Artesunate was found to suppress OVA-induced phosphorylation of Akt and its downstream signaling molecules tuberin, p70S6K and 4EBP1, and transactivation of NF-kB in lung samples. Cumulative evidence has shown that inhibitors targeting at the p70S6K and NF-kB could dampen Th2 immune responses in animal models of asthma [45,46]. Therefore, the observed reduction of IL-4, IL-5, IL-13 and eotaxin levels in BAL fluid from artesunate-treated mice may be due to inhibition of PI3K/ Akt and its downstream kinases in the inflammatory and airway resident cells. Furthermore, our in vitro study using direct OVA stimulation of lymph node cells isolated from artesunate-treated mice produced markedly lower level of IL-4, IL-5 and IL-13 together with higher IFN-c level as compared with DMSO-treated mice. These data show that the anti-inflammatory effect of artesunate is at least in part mediated through a direct suppressive action on T lymphocytes. Eosinophils play a central role in the pathogenesis of allergic inflammation [5,7]. Our present findings showed that artesunate prevented OVA-induced inflammatory cell infiltration into the airways as shown by a significant drop in total cell counts and eosinophil and lymphocyte counts in BAL fluid, and in tissue eosinophilia in lung sections. To validate the anti-inflammatory effects of artesunate in a more clinically relevant asthma model, we also observed a major reduction of total cell and eosinophil counts in BAL fluid collected from house dust mite extract-challenged- mice. Leukocyte transmigration into the airways is orchestrated by cytokines like IL-4, IL-5 and IL-13, and coordinated by specific chemokines like eotaxin and RANTES in combination with adhesion molecules such as intercellular adhesion molecule-1 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20932