Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-jB and AP-1, induced by pro-inflammatory stimuli, such as TNF-a, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-a. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity. Keywords: apocynin; NADPH oxidase; oxidative stress; asthma; inflammation; NF-kB Asthma is a chronic inflammatory lung disease characterized by infiltration of inflammatory cells, including eosinophils, and airway hyperresponsiveness (AHR). 1-5 T-helper type 2 (Th2) cells, together with other inflammatory cells such as macrophages, eosinophils, mast cells and B cells, have critical roles in the initiation, development and chronicity of this disease. 1 Upon challenge with various allergens, these inflammatory cells infiltrate into the airway and contribute to the production of Th2 cytokines, such as IL-4, IL-5 and IL-13, which are found at elevated levels in asthmatic lungs. 1-5 Th2 cytokines are pivotal for B cell maturation, IgR synthesis, airway eosinophilia, mucus secretion and ultimately AHR. Specifically, IL-4 regulates allergic inflammation by promoting Th2 cell differentiation, controlling the production of IgE in B cells, stimulating B cell proliferation, inducing the upregulation of MHC class II molecules and increasing the expression of an inducible form of the low-affinity receptor for IgE (FcRII or CD23) on B lymphocytes and macrophages. 4,5 IL-5 influences the production, maturation and activation of eosinophils. 4,5 IL-13 is a potent modulator of human monocyte and B cell function. 5 IL-13 is also capable of inducing the expression of CD23 on purified human B cells and acts as a switch factor directing IgE synthesis. 5 Increased reactive oxygen species (ROS) generation, which results in imbalance between oxidative forces and the antioxidant defense systems, has been implicated in the pathogenesis of asthma. 6-8 ROS are capable of eliciting a variety of pathological changes, including the peroxidation of lipids, proteins and DNA, and the generation of chemo-attractants, enhancement of AHR,...

Although previous studies have proposed plausible mechanisms of the activation of transforming growth factor-β-activated kinase 1 (TAK1) in inflammatory signals, including Toll-like receptors (TLRs), its activating kinase still remains to be unclear. In the present study, we have provided evidences that AMP-activated protein kinase (AMPK)-α1 has a pivotal role for activating TAK1, and thereby regulate NF-κB-dependent gene expressions in inflammatory signaling mediated by TLR4 and TNF-α stimulation. AMPK-α1 specifically interacts with TAK1 and reciprocally regulates their kinase activities. Upon the stimulation of lipopolysaccharide, AMPK-α1-knockdown (AMPK-α1KD) or TAK1-knockdown human monocytic THP-1 cells exhibit a dramatic reduction in the TAK1 or AMPK-α1 kinase activity, respectively, and subsequent suppressions of its downstream signaling cascades, which further leads to inhibitions of NF-κB and thereby productions of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Importantly, the microarray analysis of AMPK-α1KD cells revealed a dramatic reduction in the NF-κB-dependent genes induced by TLR4 and TNF-α stimulation, and the observation was in significant correlation with the results of quantitative real-time PCR. Moreover, AMPK-α1KD cells are highly sensitive to the TNF-α-induced apoptosis, which is accompanied with dramatic reductions in the NF-κB-dependent and anti-apoptotic genes. As a result, our data demonstrate that AMPK-α1 as an activating kinase of TAK1 has a key role in mediating inflammatory signals triggered by TLR4 and TNF-α.

dTransforming growth factor ␤ (TGF-␤)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-B and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1؊/؊ and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1؊/؊ mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-B-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.T ransforming growth factor ␤-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) family (1). TAK1 is essential for the production of tumor necrosis factor (TNF-␣) and other inflammatory mediators by activating several MAPKs, such as p38␣ MAPK, Jun N-terminal protein kinases 1 and 2 (JNK1 and JNK2), and extracellular signal-regulated kinases 1 and 2 (ERK1/2). TAK1 also plays a key regulatory role in several cytokine-mediated innate immunity signal transduction cascades, including interleukin-1 (IL-1) and the downstream signaling of Toll-like receptors (TLRs) and NOD1/2 (2, 3). In these pathways, various proinflammatory cytokines and TLR agonists trigger TAK1 activity, leading to its autophosphorylation and subsequent recruitment to the IB kinase (IKK) complex, ultimately resulting in the activation of the transcription factor NF-B and the upregulation of genes encoding proinflammatory cytokines, chemokines, adhesion molecules, and proteolytic enzymes.Several binding partners of TAK1, including TAK1-binding protein 1 (TAB1), TAB2, and TAB3, have been implicated in the regulation of TAK1 activity in response to various stimuli (1, 4). Previous reports demonstrated that the native forms of TAK1 comprise a catalytic kinase subunit in complex with the regulatory subunit TAB1 and either of two homologous proteins, TAB2 and TAB3 (2, 5, 6). Importantly, it has been reported that TAB1 might play a key role in the regulation of the TAK1 complex (7,8). Studies with TAB1-deficient mouse embryonic fibroblasts (TAB1 Ϫ/Ϫ MEFs) demonstrated that TAB1 is able to recruit p38␣ MAPK to the TAK1 complex f...