Artesunate Attenuates Asthma Figure 4. Effects of artesunate on OVA-induced AHR. Airway responsiveness of mechanically ventilated mice in response to aerosolized methacholine was measured 24 hours after the last saline aerosol or OVA aerosol with pretreatment of either DMSO or 30 mg/kg artesunate. AHR is expressed as percentage change from the baseline level of (A) lung resistance (Rl, n = 6 mice) and (B) dynamic compliance (Cdyn, n = 6 mice). Rl is defined as the pressure driving respiration divided by flow. Cdyn refers to the distensibility of the lung and is defined as the change in volume of the lung produced by a change in pressure across the lung. *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g004 previously [38,39]. Intratracheal administration of house dust mite extract (100 mg per mouse) on day 0, day 7 and day 14 resulted a notable increase in BAL fluid total cell and eosinophil counts, as compare with saline control. Pre-treatment with artesunate (30 mg/kg) significantly (p,0.05) suppressed the total and eosinophil counts in BAL fluid as compared with the DMSO vehicle control (Figure 7). Discussion PI3K is a family of lipid kinases comprising of 8 isoforms, of which p110d and p110c PI3Ks are enriched in leukocytes and have been shown to play a critical role in the activation, proliferation, differentiation and migration of T and B lympho- cytes, mast cells and eosinophils [18–20]. Indeed, genetic ablation of p110d or p110c PI3K in mice has been shown to dampen Th2 immune responses, eosinophilic lung infiltration, and airway hyperresponsiveness and remodeling [21–23]. Therapeutic strat- egies targeted at the PI3K/Akt signaling pathway such as using Figure 5. Effects of artesunate on OVA-induced inflammatory gene expression, PI3K/Akt activation and NF-kB DNA-binding activity in allergic airway inflammation. (A) Lung tissues were collected 24 hours after the last OVA aerosol challenge. Total mRNA was extracted using TriZol reagent and the quantitative real time PCR was performed. All reactions were run in triplicate and three independent experiments for each target. The relative quantity of target gene expression was automatically normalized by GADPH as an internal control and values shown were the ratios of various treatments to saline group. (B) Immunoblotting of Akt, tuberin, p70S6K and 4E-BP1 in protein extracts of lung tissues isolated from mice 24 hours after the last saline aerosol or OVA aerosol challenge pretreated with either DMSO or 30 mg/kg artesunate. b-actin was used as an internal control. The experiments were repeated for three times (n = 3 mice) with similar pattern of results. (C) Nuclear p65 DNA-binding activity was determined using a TransAMTM p65 transcription factor ELISA kit. Values shown are the mean 6 SEM of four separate experiments. *Significant difference from DMSO control, p,0.05. doi:10.1371/journal.pone.0020932.g005 adenovirus-carrying phosphatase and tensin homologue deleted on chromosome ten (PTEN) cDNA [40], dominant-negative class IA PI3K-TAT fusion protein [41], and PI3K non-selective and isoform-selective small molecule inhibitors [42,43,44] have demonstrated beneficial effects in experimental asthma models. Our findings reveal for the first time significant inhibition of Akt(ser473) phosphorylation and of its downstream kinases by artesunate in both OVA-challenged lungs in vivo and EGF- stimulated normal human bronchial epithelial cells in vitro. Taken together, we have established that anti-malarial drug artesunate, a semi-synthetic derivative of artemisinin isolated from the herb Artemisia annua, can effectively suppress various aspects of OVA- induced Th2-mediated allergic airway inflammation in mice via inhibition of the PI3K/Akt signaling pathway. PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20932