Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vitro and in vivo. However, the molecular mechanism of luteolin-mediated immune modulation has not been fully understood. In this study, we examined the effects of luteolin on the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)), as well as the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in mouse alveolar macrophage MH-S and peripheral macrophage RAW 264.7 cells. Luteolin dose-dependently inhibited the expression and production of these inflammatory genes and mediators in macrophages stimulated with lipopolysaccharide (LPS). Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay further confirmed the suppression of LPS-induced TNF- alpha, IL-6, iNOS and COX-2 gene expression by luteolin at a transcriptional level. Luteolin also reduced the DNA binding activity of nuclear factor-kappa B (NF-kappaB) in LPS-activated macrophages. Moreover, luteolin blocked the degradation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 subunit. In addition, luteolin significantly inhibited the LPS-induced DNA binding activity of activating protein-1 (AP-1). We also found that luteolin attenuated the LPS-mediated protein kinase B (Akt) and IKK phosphorylation, as well as reactive oxygen species (ROS) production. In sum, these data suggest that, by blocking NF-kappaB and AP-1 activation, luteolin acts to suppress the LPS-elicited inflammatory events in mouse alveolar macrophages, and this effect was mediated, at least in part, by inhibiting the generation of reactive oxygen species. Our observations suggest a possible therapeutic application of this agent for treating inflammatory disorders in lung.

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.Matriptase, a type II transmembrane serine protease, plays essential roles in epidermal differentiation and barrier function (1, 2). Mutations of matriptase are associated with autosomal recessive ichthyosis and hypotrichosis (3). Four mutations have been identified in ST14, the gene that encodes for matriptase (3-5). Targeted deletion of matriptase in mice also causes severe epidermal defects with impairment of desquamation, lipid matrix formation, and profilaggrin processing. These defects lead to compromised epidermal barrier function and postnatal death (2). The severe epidermal defects appear to result from the lack of activation of the glycosylphosphatidylinositolanchored serine protease prostasin by matriptase, because both matriptase-deficient and prostasin-deficient mice share an almost identical pattern of epidermal defects. Furthermore, lack of prostasin activation was observed in matriptase-deficient mouse skin (6, 7), suggesting that much of the function of matriptase in the epidermis is manifested through the activation of prostasin.Hepatocyte growth factor activator inhibitor-1 (HAI-1) 2 is a transmembrane Kunitz-type serine protease inhibitor (8). HAI-1 can inhibit matriptase in a competitive and reversible manner, consistent with the characteristics of Kunitz-type serine protease inhibitors (...

Lonicera japonica (Caprifoliaceae) has been known as an anti-inflammatory herb in traditional Chinese medicine for thousands of years and is used constantly for upper respiratory tract infections. Luteolin, an active flavonoid compound isolated from Lonicera japonica, has a spectrum of biological activities, especially with antioxidative and anti-inflammatory properties. However, whether luteolin has a direct inhibitory effect on lung fibrosis has not been established. In this study, we examined the effects of luteolin on lung fibrosis both in vivo and in vitro. We found that oral administration of luteolin (10 mg/kg) efficiently suppressed the neutrophil infiltration as well as TNF-α and IL-6 elevation in the bronchoalveolar lavage fluid in bleomycin-instilled C57BL/6J mice. Luteolin also alleviated collagen deposition, TGF-β1 expression, and lung fibrosis upon bleomycin instillation. A similar tendency was observed in both early and delayed luteolin-treated groups. Next, our in vitro studies showed that luteolin inhibited TGF-β1-induced α-SMA, type I collagen, and vimentin expression in primary cultured mouse lung fibroblasts. Moreover, luteolin significantly blocked TGF-β1-mediated epithelial marker (E-cadherin) downregulation and mesenchymal cell markers (fibronectin and vimentin) upregulation, as well as retaining epithelial morphology in human alveolar epithelial-derived A549 cells. Additionally, luteolin could attenuate TGF-β1-induced Smad3 phosphorylation in both lung fibroblasts and A549 cells. These findings suggest that luteolin has a potent antifibrotic activity; this effect was mediated, at least in part, by inhibition of lung inflammation and suppression of myofibroblast differentiation as well as epithelial-to-mesenchymal transition.

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Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by the activation of fibroblasts and the overproduction of extracellular matrix. Fibroblast resistance to apoptosis leads to increased fibrosis. Targeting fibroblasts with apoptotic agents represents a major therapeutic intervention for debilitating IPF. Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. However, the effects of gallic acid on lung fibroblasts have not been investigated. The aim of the present study is to determine the effects of gallic acid on primary cultured mouse fibroblasts. Our results showed that gallic acid induces the apoptotic death of fibroblasts via both intrinsic and extrinsic apoptotic pathways by the elevation of PUMA, Fas, and FasL protein levels. Moreover, intracellular reactive oxygen species (ROS) generation and 8-hydroxy-2'-deoxyguanosine production were observed in gallic acid-stimulated fibroblasts. Mechanistic studies showed that gallic acid induces early phosphorylation of p53(Ser18) and histone 2AX(Ser139) (H2AX) via ataxia telangiectasia mutated (ATM) activation in response to ROS-provoked DNA damage. When mouse lung fibroblasts were treated with caffeine, an ATM kinase inhibitor, the levels of p53, phosphorylated p53(Ser18), and cell death induced by gallic acid were significantly attenuated. Additionally, pretreatment with antioxidants drastically inhibited the gallic acid-induced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation and phosphorylation of p53(Ser18) and ATM(Ser1981), as well as apoptosis. Our results provide the first evidence of the activation of ROS-dependent ATM/p53 signaling as a critical mechanism of gallic acid-induced cell death in primary cultured mouse lung fibroblasts.