Receptor-interacting protein 2 gene silencing attenuates allergic airway inflammation.

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-1ß, 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.

Acute treatment with bone marrow-derived mononuclear cells attenuates the organ injury/dysfunction induced by hemorrhagic shock in the rat.

Recent evidence suggests that cell therapy such as the injection of bone marrow-derived mononuclear cells (BMMNCs) can exert protective effects in various conditions associated with ischemia-reperfusion injury. Here, we investigate the effects of BMMNCs on the organ injury/dysfunction induced by hemorrhagic shock (HS). Thirty-seven anesthetized male Wistar rats were subjected to hemorrhage by reducing mean arterial pressure to 35 ± 5 mmHg for 90 min, followed by resuscitation with 20 mL/kg Ringer's lactate administered over 10 min and 50% of the shed blood over 50 min. Rats were killed 4 h after the onset of resuscitation. Bone marrow-derived mononuclear cells were freshly isolated from rat tibias and femurs using Percoll density gradient centrifugation, and BMMNCs (1 × 10 cells per rat in 1 mL/kg phosphate-buffered saline, i.v.) were administered on resuscitation. Hemorrhagic shock resulted in significant organ injury/dysfunction (renal, hepatic, neuromuscular) and inflammation (hepatic, lung). In rats subjected to HS, administration of BMMNCs significantly attenuated (i) organ injury/dysfunction (renal, hepatic, neuromuscular) and inflammation (hepatic, lung), (ii) increased the phosphorylation of Akt and glycogen synthase kinase-3ß, (iii) attenuated the activation of nuclear factor-κB, (iv) attenuated the increase in extracellular signal-regulated kinase 1/2 phosphorylation, and (v) attenuated the increase in expression of intercellular adhesion molecule-1. Our findings suggest that administration of BMMNCs protects against the induction of early organ injury/dysfunction caused by severe HS by a mechanism that may involve activation of Akt and the inhibition of glycogen synthase kinase-3ß and nuclear factor-κB.

Fisetin, a bioactive flavonol, attenuates allergic airway inflammation through negative regulation of NF-κB.

Persistent activation of nuclear factor-κB (NF-κB) has been associated with the development of asthma. Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally occurring bioactive flavonol, has been shown to inhibit NF-κB activity. We hypothesized that fisetin may attenuate allergic asthma via negative regulation of the NF-κB activity. Female BALB/c mice sensitized and challenged with ovalbumin 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. Fisetin dose-dependently inhibited ovalbumin-induced increases in total cell count, eosinophil count, and IL-4, IL-5 and IL-13 levels recovered in bronchoalveolar lavage fluid. It attenuated ovalbumin-induced lung tissue eosinophilia and airway mucus production, mRNA expression of adhesion molecules, chitinase, IL-17, IL-33, Muc5ac and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. Fisetin blocked NF-κB subunit p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of ovalbumin-challenged mice. In normal human bronchial epithelial cells, fisetin repressed TNF-α-induced NF-κB-dependent reporter gene expression. Our findings implicate a potential therapeutic value of fisetin in the treatment of asthma through negative regulation of NF-κB pathway.

Anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3-kinase/Akt pathway.

BACKGROUND: Phosphoinositide 3-kinase (PI3K)/Akt pathway is linked to the development of asthma. Anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua, 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

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-3ß 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-3ß and NF-κB.

A novel anti-apoptotic role for apolipoprotein L2 in IFN-γ-induced cytotoxicity in human bronchial epithelial cells.

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN-γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN-γ-stimulated normal human bronchial epithelial cells (HBEC) using a 2-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS. IFN-γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN-γ, in lung tissues from an IFN-γ-predominant influenza A virus-infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN-γ-induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN-γ-induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub-G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN-γ-induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide-double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase-dependent manner. Our results reveal a novel function for ApoL2 in conferring anti-apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN-γ, in maintaining airway epithelial layer integrity.