Artesunate Attenuates Asthma Figure 1. Effects of artesunate on OVA-induced inflammatory cell recruitment and mucus hypersecretion. (A) Inflammatory cell counts in BAL fluid obtained from sensitized mice 24 hours after the last saline aerosol (n = 7 mice) or OVA aerosol (n = 9 mice) challenge. Artesunate dose- dependently reduced OVA-induced inflammatory cell counts in BAL fluid from sensitized mice 24 hours after the last OVA aerosol challenge (DMSO, n = 9; 3 mg/kg, n = 7; 10 mg/kg, n = 9; and 30 mg/kg, n = 10). Differential cell counts were performed on a minimum of 500 cells to identify eosinophil (Eos), macrophage (Mac), neutrophil (Neu), and lymphocyte (Lym). Histological sections of lung tissue eosinophilia (B, magnification6200) and mucus secretion (C, magnification6200) 24 hours after the last challenge of saline aerosol, OVA aerosol, OVA aerosol plus DMSO, or OVA aerosol plus 30 mg/ kg artesunate were evaluated. Quantitative analyses of inflammatory cell infiltration (D) and mucus production (E) in lung sections were performed as previously described [56]. Scoring of inflammatory cells and goblet cells was performed in at least 3 different fields for each lung section. Mean scores were obtained from 4 animals. *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g001 IL-33, AMCase, Ym-2, YKL-40, ICAM-1, VCAM-1 and E- selectin, in the allergic airways (Figure 5A). To verify that the anti-inflammatory mechanism of action by artesunate in OVA-challenged mice was mediated through the inhibition of the PI3K/Akt signaling pathway, we examined the phosphorylation cascade of Akt, tuberin, p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in lung tissues obtained 24 hours after the last OVA or saline aerosol challenge. OVA challenge markedly raised the phosphorylation state of Akt(ser473), tuberin(thr1462), p70S6K(thr389) and 4E-BP1(ser65) as compared with saline aerosol control (Figure 5B). Artesunate (30 mg/kg) markedly reduced the phsophorylation of Akt, tuberin, p70S6K and 4E-BP1 to the basal levels. Besides, PI3K/Akt pathway activation has been shown to promote NF-kB DNA binding activity [34]. Artesunate signifi- cantly suppressed OVA-induced NF-kB transactivation in lung tissues to basal level (Figure 5C). Our findings suggest that artesunate may exert its anti-inflammatory actions via inhibition of PI3K/Akt pathway. Artesunate inhibits EGF-induced PI3K/Akt activation in primary human bronchial epithelial cells To further explore anti-inflammatory mechanisms of action of artesunate in a relevant human airway cell type, we studied the effects of artesunate on epidermal growth factor (EGF)-induced activation of PI3K/Akt signaling pathway and cytokine mRNA PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20932