Anti-Malarial Drug Artesunate Attenuates Experimental Allergic Asthma via Inhibition of the Phosphoinositide 3-Kinase/Akt Pathway

, and has been shown to inhibit PI3K/Akt activity. We hypothesized that artesunate may attenuate allergic asthma via inhibition of the PI3K/Akt signaling pathway.Female BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Artesunate dose-dependently inhibited OVA-induced increases in total and eosinophil counts, IL-4, IL-5, IL-13 and eotaxin levels in bronchoalveolar lavage fluid. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, IL-17, IL-33 and Muc5ac in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, artesunate blocked epidermal growth factor-induced phosphorylation of Akt and its downstream substrates tuberin, p70S6 kinase and 4E-binding protein 1, and transactivation of NF-κB. Similarly, artesunate blocked the phosphorylation of Akt and its downstream substrates in lung tissues from OVA-challenged mice. Anti-inflammatory effect of artesunate was further confirmed in a house dust mite mouse asthma model.Artesunate ameliorates experimental allergic airway inflammation probably via negative regulation of PI3K/Akt pathway and the downstream NF-κB activity. These findings provide a novel therapeutic value for artesunate in the treatment of allergic asthma

Receptor-Interacting Protein 2 Gene Silencing Attenuates Allergic Airway Inflammation

Abstract Persistent activation of NF-κB has been associated with the development of asthma. Receptor-interacting protein 2 (Rip2) is a transcriptional product of NF-κB activation. It is an adaptor protein with serine/threonine kinase activity and has been shown to positively regulate NF-κB activity. We investigated potential protective effects of Rip2 gene silencing using small interfering RNA (siRNA) in an OVA-induced mouse asthma model. Rip2 protein level was found to be upregulated in allergic airway inflammation. A potent and selective Rip2 siRNA given intratracheally knocked down Rip2 expression in OVA-challenged lungs and reduced OVA-induced increases in total and eosinophil counts, and IL-4, IL-5, IL-13, IL-1β, IL-33, and eotaxin levels in bronchoalveolar lavage fluid. Rip2 silencing blocked OVA-induced inflammatory cell infiltration and mucus hypersecretion as observed in lung sections, and mRNA expression of ICAM-1, VCAM-1, E-selectin, RANTES, IL-17, IL-33, thymic stromal lymphopoietin, inducible NO synthase, and MUC5ac in lung tissues. In addition, elevation of serum OVA-specific IgE level in mouse asthma model was markedly suppressed by Rip2 siRNA, together with reduced IL-4, IL-5, and IL-13 production in lymph node cultures. Furthermore, Rip2 siRNA-treated mice produced significantly less airway hyperresponsiveness induced by methacholine. Mechanistically, Rip2 siRNA was found to enhance cytosolic level of IκBα and block p65 nuclear translocation and DNA-binding activity in lung tissues from OVA-challenged mice. Taken together, our findings clearly show that knockdown of Rip2 by gene silencing ameliorates experimental allergic airway inflammation, probably via interruption of NF-κB activity, confirming Rip2 a novel therapeutic target for the treatment of allergic asthma.</jats:p

Effects of artesunate on EGF stimulation of normal human bronchial epithelial cells.

<p>(A) Epithelial cells were stimulated with 100 ng/ml EGF in the presence and absence of 10 µM artesunate for 5, 15 and 30 minutes before total proteins were extracted for subsequent immunoblotting analysis. β-actin was used as an internal control. (B) DNA-binding activity of p65 NF-κB in nuclear extracts of epithelial cells stimulated with EGF for 30 minutes in the presence and absence of 10 µM artesunate was determined using a TransAM™ p65 transcription factor ELISA kit. (C) Epithelial cells were stimulated with 100 ng/ml EGF in the presence and absence of 10 µM 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. β-actin was used as an internal control. Representative gels from 4 separate experiments with similar pattern of results. Values shown are the mean ± SEM of three separate experiments. *Significant difference from DMSO control, p<0.05.</p

Effects of artesunate on OVA-induced inflammatory cell recruitment and mucus hypersecretion.

<p>(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, magnification×200) and mucus secretion (C, magnification×200) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020932#pone.0020932-Zhou2" target="_blank">[56]</a>. 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.</p

Effects of artesunate on OVA-induced BAL fluid cytokine and chemokine levels.

<p>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-γ 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-γ at 15.6 pg/ml; and eotaxin at 2 pg/ml. Values shown are the mean ± SEM. *Significant difference from DMSO control, p<0.05.</p

Effects of artesunate on house dust mite extracts-induced pulmonary inflammatory cell recruitment.

<p>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.</p

Effects of artesunate on OVA-induced inflammatory gene expression, PI3K/Akt activation and NF-κB DNA-binding activity in allergic airway inflammation.

<p>(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. β-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 TransAM™ p65 transcription factor ELISA kit. Values shown are the mean ± SEM of four separate experiments. *Significant difference from DMSO control, p<0.05.</p

A Nonerythropoietic Peptide that Mimics the 3D Structure of Erythropoietin Reduces Organ Injury/Dysfunction and Inflammation in Experimental Hemorrhagic Shock

Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect—an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB