Artesunate Attenuates Asthma Figure 2. Effects of artesunate on OVA-induced BAL fluid cytokine and chemokine levels. BAL fluids were collected 24 hours after the last OVA aerosol challenge. Levels of IL-4, IL-5, IL-12, IL-13, eotaxin and IFN-c were analyzed using ELISA (n = 6–9 mice). Lower limits of detection were as follows: IL-4 and IL-5 at 4 pg/ml; IL-12, IL-13 and IFN-c at 15.6 pg/ml; and eotaxin at 2 pg/ml. Values shown are the mean 6 SEM. *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g002 expression in normal human bronchial epithelial cells. EGF plays a critical role in asthma [35,36] and is a potent stimulator of human airway epithelial cells [37]. EGF induced a rapid phosphorylation of Akt, tuberin, p70S6K and 4E-BP1 (Figure 6A). This was accompanied with the up-regulation of NF-kB DNA binding activity (Figure 6B). Artesunate markedly inhibited the EGF-induced phosphorylation of Akt, tuberin, p70S6K and 4E-BP1, and NF-kB transactivation. Furthermore, artesunate noticeably blocked EGF-induced up-regulation of IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and RANTES mRNA expression in human bronchial epithelial cells (Figure 6C). MTT assay was conducted to examine potential cytotoxicity of artesunate on normal human bronchial epithelial cells. No cytotoxicity on bronchial epithelial cells was observed with artesunate drug concentrations (0.1–100 mM) used in the in vitro experiments (data not shown). Artesunate suppresses airway inflammation in a house dust mite extract-induced asthma model To validate the anti-inflammatory effects of artesunate in airway inflammation more comparable to human situation, we have developed a house dust mite mouse asthma model as described Figure 3. Effects of artesunate on OVA-specific lymphocyte response in vitro. Thoracic lymph nodes cells (n = 4 mice) were harvested from mice 24 hours after the last OVA or saline aerosol challenge, and cultured for 72 hours with medium alone or OVA (200 mg/ml). The levels of IL-4, IL-5, IL-13 and IFN-c in culture supernatant were determined using ELISA. Values shown are the mean 6 SEM of triplicate cultures of pooled lymph node cell suspensions. *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g003 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20932