Se Eun Byeon

The Role of Src Kinase in Macrophage-Mediated Inflammatory Responses

Src kinase (Src) is a tyrosine protein kinase that regulates cellular metabolism, survival, and proliferation. Many studies have shown that Src plays multiple roles in macrophage-mediated innate immunity, such as phagocytosis, the production of inflammatory cytokines/mediators, and the induction of cellular migration, which strongly implies that Src plays a pivotal role in the functional activation of macrophages. Macrophages are involved in a variety of immune responses and in inflammatory diseases including rheumatoid arthritis, atherosclerosis, diabetes, obesity, cancer, and osteoporosis. Previous studies have suggested roles for Src in macrophage-mediated inflammatory responses; however, recently, new functions for Src have been reported, implying that Src functions in macrophage-mediated inflammatory responses that have not been described. In this paper, we discuss recent studies regarding a number of these newly defined functions of Src in macrophage-mediated inflammatory responses. Moreover, we discuss the feasibility of Src as a target for the development of new pharmaceutical drugs to treat macrophage-mediated inflammatory diseases. We provide insights into recent reports regarding new functions for Src that are related to macrophage-related inflammatory responses and the development of novel Src inhibitors with strong immunosuppressive and anti-inflammatory properties, which could be applied to various macrophage-mediated inflammatory diseases.

In vitro and in vivo anti-inflammatory effects of taheebo, a water extract from the inner bark of Tabebuia avellanedae.

AIM OF STUDY Tabebuia spp. (Bignoniaceae) are native to tropical rain forests throughout Central and South America and have long been used as a folk medicine to treat bacterial infection, blood coagulation, cancer and inflammatory diseases. In this study, we aimed to demonstrate the ethnopharmacological activity of Tabebuia avellanedae in various in vitro and in vivo inflammatory conditions. MATERIALS AND METHODS To do this, LPS-stimulated macrophages and arachidonic acid or croton oil-induced mouse ear edema models were employed. RESULTS The water extract (taheebo) of Tabebuia avellanedae significantly suppressed the production of prostaglandin (PG) E(2) and nitric oxide (NO), and blocked the mRNA expression of their catalyzing enzymes (cyclooxygenase [COX)-II] and inducible NO synthase [iNOS], respectively), in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The blockade of inflammatory mediators by taheebo seemed to be the result of the interruption of extracellular signal-related kinase (ERK) activation, according to immunoblotting analysis and the NO assay, where LPS strongly induced the phosphorylation (a hallmark of activation) of ERK, and U0126, a selective ERK inhibitor, was found to strongly inhibit PGE(2) production. Similarly, oral administration of taheebo (100mg/kg) for 1 week completely diminished mouse ear edema induced by arachidonic acid, an activator of COX-II, but not croton oil, an activator of lipoxygenase. CONCLUSIONS These data suggest that the ethnopharmacological action of taheebo may be due to its negative modulation of macrophage-mediated inflammatory responses by suppressing PGE(2) production. Thus, this water extract may be developed as a new therapeutic remedy for various inflammatory diseases such as arthritis and atherosclerosis.

Molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from Korean red ginseng.

Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng, displays immunostimulatory and antitumor activities. Even though numerous studies have been reported, the mechanism as to how RGAP is able to stimulate the immune response is not clear. In this study, we aimed to explore the mechanism of molecular activation of RGAP in macrophages. RGAP treatment strongly induced NO production in RAW264.7 cells without altering morphological changes, although the activity was not strong compared to LPS-induced dendritic-like morphology in RAW264.7 cells. RGAP-induced NO production was accompanied with enhanced mRNA levels of iNOS and increases in nuclear transcription factors such as NF-κB, AP-1, STAT-1, ATF-2, and CREB. According to pharmacological evaluation with specific enzyme inhibitors, Western blot analysis of intracellular signaling proteins and inhibitory pattern using blocking antibodies, ERK, and JNK were found to be the most important signaling enzymes compared to LPS signaling cascade. Further, TLR2 seems to be a target surface receptor of RGAP. Lastly, macrophages isolated from RGS2 knockout mice or wortmannin exposure strongly upregulated RGAP-treated NO production. Therefore, our results suggest that RGAP can activate macrophage function through activation of transcription factors such as NF-κB and AP-1 and their upstream signaling enzymes such as ERK and JNK.

In vitro and in vivo anti-inflammatory activities of Polygonum hydropiper methanol extract.

ETHNOPHARMACOLOGICAL RELEVANCE Polygonum hydropiper L. (Polygonaceae) has been traditionally used to treat various inflammatory diseases such as rheumatoid arthritis. However, no systematic studies on the anti-inflammatory actions of Polygonum hydropiper and its inhibitory mechanisms have been reported. This study is therefore aimed at exploring the anti-inflammatory effects of 99% methanol extracts (Ph-ME) of this plant. MATERIALS AND METHODS The effects of Ph-ME on the production of inflammatory mediators in RAW264.7 cells and peritoneal macrophages were investigated. Molecular mechanisms underlying the effects, especially inhibitory effects, were elucidated by analyzing the activation of transcription factors and their upstream signalling, and by evaluating the kinase activities of target enzymes. Additionally, a dextran sulphate sodium (DSS)-induced colitis model was employed to see whether this extract can be used as an orally available drug. RESULTS Ph-ME dose-dependently suppressed the release of nitric oxide (NO), tumour necrosis factor (TNF)-α, and prostaglandin (PG)E(2), in RAW264.7 cells and peritoneal macrophages stimulated by lipopolysaccharide (LPS). Ph-ME inhibited mRNA expression of pro-inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and TNF-α by suppressing the activation of nuclear factor (NF)-κB, activator protein (AP-1), and cAMP responsive element binding protein (CREB), and simultaneously inhibited its upstream inflammatory signalling cascades, including cascades involving Syk, Src, and IRAK1. Consistent with these findings, the extract strongly suppressed the kinase activities of Src and Syk. Based on HPLC analysis, quercetin, which inhibits NO and PGE(2) activities, was found as one of the active ingredients in Ph-ME. CONCLUSION Ph-ME exerts strong anti-inflammatory activity by suppressing Src/Syk/NF-κB and IRAK/AP-1/CREB pathways, which contribute to its major ethno-pharmacological role as an anti-gastritis remedy.

Src-mediated regulation of inflammatory responses by actin polymerization.

Although the role of the actin cytoskeleton has become increasingly elucidated, the role of actin polymerization in inflammatory processes remains poorly understood. Here, we examine the role of the actin cytoskeleton during LPS-mediated inflammatory events in RAW264.7 cells and peritoneal macrophages. We observed that actin cytoskeleton disruption by cytochalasin B and siRNA to cytoplasmic actin strongly down-regulated LPS-mediated inflammatory responses such as NO production, PGE(2) release, and TNF-alpha secretion. Actin cytoskeleton disruption by cytochalasin B down-regulated a series of signaling cascades including PI3K, Akt, and IKK, but not MAPKs, necessary for NF-kappaB activation without down-regulating total forms of the proteins as assessed by measuring their phosphorylation levels. In particular, cytochalasin B significantly inhibited LPS-induced both phosphorylation and kinase activity of Src without altering total level, implying that Src may be a potential pharmacological target of actin cytoskeleton rearrangement. Moreover, the direct association of Src with actin was actin polymerization-dependent according to immunoprecipitation analysis performed with a GFP-actin wild type and HA-tagged Src. Therefore, our data suggest that actin cytoskeleton rearrangement may be a key event during the regulation of inflammatory responses that modulates the activity of Src and its downstream signaling molecules.

In vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmentosum.

AIM OF STUDY Acer tegmentosum has been traditionally used for folk medicine to treat hepatic disorders such as hepatitis, hepatic cancer, and hepatic cirrhosis. In this study, we demonstrate the ethno-pharmacological activity of Acer tegmentosum in in vitro and in vivo inflammatory conditions. RESULTS The 70% ethanol extract (At-EE) of Acer tegmentosum dose-dependently diminished the production of nitric oxide (NO), tumour necrosis factor (TNF)-alpha, and prostaglandin (PG)E(2), in lipopolysaccharide (LPS)-activated RAW264.7 cells and peritoneal macrophages, by a transcriptional mechanism. At-EE also suppressed the activation of nuclear factor (NF)-kappaB, activator protein (AP)-1, and cAMP-responsive element binding (CREB), and simultaneously blocked their upstream inflammatory signalling cascades, including Akt, p38, and JNK. Furthermore, At-EE protected against LPS-induced cell death induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) and neutralized reactive species generation. In agreement with the in vitro results, orally administered At-EE strongly ameliorated ear oedema formation induced by arachidonic acid. CONCLUSION At-EE displays strong anti-inflammatory activities in vitro and in vivo, contributing to its major ethno-pharmacological role such as anti-hepatitis remedy and may be applicable to novel anti-inflammatory therapeutics.

Inhibitory effect of saponin fraction from Codonopsis lanceolata on immune cell-mediated inflammatory responses.

Saponin components are known to be pharmaceutically, cosmetically and nutraceutically valuable principles found in various herbal medicine. In this study, we evaluated the inhibitory role of saponin fraction (SF), prepared from C. lanceolata, an ethnopharmacologically famous plant, on various inflammatory responses managed by monocytes, macrophages, lymphocytes and mast cells. SF clearly suppressed the release of nitric oxide (NO) and tumor necrosis factor (TNF)-α, but not prostaglandin E2 (PGE2). While this fraction did not scavenge the reactivity of SNP-induced radicals in RAW264. 7 cells, it negatively modulated the phagocytic uptake of macrophages treated with FITC-dextran. Interestingly, SF completely diminished cell-cell adhesion events induced by both CD29 and CD43, but not cell-fibronectin adhesion. Concanavalin (Con) A [as well phytohemaglutinin A (PHA)]-induced proliferation of splenic lymphocytes as well as interferon (IFN)-γ production were also clearly suppressed by SF treatment. Finally, SF also significantly blocked the degranulation process of mast cell line RBL-2H3 cell as assessed by DNP-BSA-induced β-hexosaminidase activity. The anti-inflammatory activities of SF on NO production seemed to be due to inhibition of nuclear factor (NF)-κB activation signaling, since it blocked the phosphorylation of inhibitor of κB (IκB)α as well as inducible NO synthase (iNOS) expression. Therefore, these results suggest that SF may be considered as a promising herbal medicine with potent anti-inflammatory actions.

Hydroquinone regulates hemeoxygenase-1 expression via modulation of Src kinase activity through thiolation of cysteine residues.

The hydroxylated benzene metabolite hydroquinone (HQ) is mainly generated from benzene, an important industrial chemical, and is also a common dietary component. Although numerous papers have addressed the potential role of HQ in tumorigenic responses, the immunosuppressive and anti-inflammatory effects of hydroquinone have also been considered. In this study, we characterized the mechanism of the induction of hemeoxygenase (HO)-1 and other phase 2 enzymes by HQ and its derivatives. HQ upregulated the mRNA and protein levels of HO-1 by increasing the antioxidant-response element-dependent transcriptional activation of Nrf-2. Src knockdown or deficiency induced via siRNA treatment and infection with a retrovirus expressing shRNA targeting Src, as well as exposure to PP2, a Src kinase inhibitor, strongly abrogated HO-1 expression. Interestingly, HQ directly targeted and bound to the sulfhydryl group of cysteine-483 (C483) and C400 residues of Src, potentially leading to disruption of intracellular disulfide bonds. Src kinase activity was dramatically enhanced by mutation of these cysteine sites, implying that these sites may play an important role in the regulation of Src kinase activity. Therefore, our data suggest that Src and, particularly, its C483 target site can be considered as prime molecular targets of the HQ-mediated induction of phase 2 enzymes, which is potentially linked to HO-1-mediated cellular responses such as immunosuppressive and anti-inflammatory actions.

3-(4-(tert-Octyl)phenoxy)propane-1,2-diol suppresses inflammatory responses via inhibition of multiple kinases

Novel anti-inflammatory compounds were synthesised by derivatization of militarin, a compound isolated from Cordyceps militaris that is an ethnopharmacologically well-known herbal medicine with multiple benefits such as anti-cancer, anti-inflammatory, anti-obesity, and anti-diabetic properties. In this study, we explored the in vitro and in vivo anti-inflammatory potencies of these compounds during inflammatory responses, their inhibitory mechanisms, and acute toxicity profiles. To do this, we studied inflammatory conditions using in vitro lipopolysaccharide-treated macrophages and several in vivo inflammatory models such as dextran sodium sulphate (DSS)-induced colitis, EtOH/HCl-induced gastritis, and arachidonic acid-induced ear oedema. Methods used included real-time PCR, immunoblotting analysis, immunoprecipitation, reporter gene assays, and direct kinase assays. Of the tested compounds, compound III showed the highest nitric oxide (NO) inhibitory activity. This compound also inhibited the production of prostaglandin (PG)E(2) at the transcriptional level by suppression of Syk/NF-κB, IKKɛ/IRF-3, and p38/AP-1 pathways in lipopolysaccharide (LPS)-activated RAW264.7 cells and peritoneal macrophages. Consistent with these findings, compound III strongly ameliorated inflammatory symptoms in colitis, gastritis, and ear oedema models. In acute toxicity tests, there were no significant differences in body and organ weights, serum parameters, and stomach lesions between the untreated and compound III-treated mice. Therefore, this compound has the potential to be served as a lead chemical for developing a promising anti-inflammatory drug candidate with multiple kinase targets.

Inhibition of 2,4-dinitrofluorobenzene-induced atopic dermatitis by topical application of the butanol extract of Cordyceps bassiana in NC/Nga mice

ETHNOPHARMACOLOGICAL SIGNIFICANCE The Cordyceps species are insect-borne mushrooms that have been ethnopharmacologically used for skin diseases such as eczema and dermatitis. AIM OF THE STUDY In this study, we investigated the curative effects of the butanol fraction (CBBF) of Cordyceps bassiana on atopic dermatitis. MATERIALS AND METHODS Dermatitis was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB) in NC/Nga mice. After a topical application of CBBF on the skin lesions, the dermatitis score, epidermal thickness, mast cell number, and interleukin (IL)-4 and interferon (IFN)-γ, as well as the levels of histamine and immunoglobulin E (IgE) in the serum, were measured. Moreover, effect of CBBF on histamine release was examined using RBL-2H3 under stimulation with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). RESULTS CBBF inhibited atopic dermatitis symptoms and signs in the DNFB-treated NC/Nga mice. The suppressive activity of topically applied CBBF may be due to the dose-dependent blockade of a series of immunopathological events, including the release of histamine, the production of IgE, and the secretion of IL-4 and IFN-γ. However, this extract did not directly suppress the degranulation process, assessed by measuring β-hexosaminidase release. CONCLUSIONS Our results suggest that CBBF can be applied as an effective herbal remedy to treat atopic dermatitis.