Anti-inflammatory activity of Motherwort (Leonurus sibiricus L.)

Motherwort (MW), a Korean folk medicine, has been applied to treat inflammatory disease. However, its effect on inflammatory cytokine release from mast cells is not well known. We investigated the anti- inflammatory effect of MW on the secretion of inflammatory cytokine such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 and IL-8 in human mast cell line (HMC-1). MW was treated in vitro before activation of HMC-1 cells with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187. MW had no cytotoxic effects on HMC-1 cell viability. MW (1?mg/ml) inhibited PMA plus A23187-stimulated gene expression and production of TNF-α, IL-6, and IL-8. Stimulation with PMA plus A23187 induced NF-κB activation in HMC-1 cells, which was inhibited by MW (1?mg/ml). MW inhibited secretion of TNF-α, IL-6, and IL-8 possibly by inhibiting NF-κB activation. These results indicate that MW may be helpful in regulating inflammatory diseases.     

Apigenin inhibits release of inflammatory mediators by blocking the NF-κB activation pathways in the HMC-1 cells

Apigenin is a plant flavonoid and a pharmacologically active agent that has been isolated from several plant species. However, the molecular mechanism of apigenin-mediated immune modulation has not been fully understood. One of the possible mechanisms of its protective effects is the down-regulation of inflammatory responses. In this study, we used cells from the human mast cell line (HMC-1) to investigate this effect. Apigenin significantly inhibits the inductive effect of phorbol 12-myristate 13-acetate (PMA) plus A23187 on the production of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-8, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, apigenin attenuated the cyclooxygenase (COX)-2 expression and intracellular Ca(2+) level. In activated HMC-1 cells, apigenin inhibited the PMA plus A23187-induced activation of nuclear factor (NF)-κB, IκB degradation, and luciferase activity. Furthermore, apigenin suppressed the expression of TNF-α, IL-8, IL-6, GM-CSF, and COX-2 by decreasing the intracellular Ca(2+) level and inhibiting NF-κB activation. These results indicate that apigenin has a potential regulatory effect on inflammatory reactions that are mediated by mast cells.     

The Effect of SHJKS on Cytokines Production and NF-κB Activation in the Peripheral Blood Mononuclear Cells of Patients with Cerebral Infarction

The Korean genuine medicine “Seonghyangjeongkisan” (SHJKS) has long been used for various cerebrovascular diseases. However, very little scientific investigation has been carried out. Cytokines involved in the regulation of inflammatory reactions and immune responses may play a role in the pathogenesis of cerebral infarction (CI). The aim of the present study is to elucidate how SHJKS modulates the inflammatory reaction in lipopolysaccaride (LPS) plus phytohaemagglutinin (PHA)-stimulated peripheral mononuclear cells (PBMCs) from CI patients. The amount of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 in PBMC culture supernatant was significantly increased in the LPS plus PHA treated cells compared to unstimulated cells. SHJKS inhibited the TNF-α, IL-1β, IL-6, and IL-8 production in dose dependent manner. Maximal inhibition rate of the TNF-α, IL-1β, IL-6, and IL-8 by SHJGS (1.0 mg/ml) was 68.01 ± 0.28% (P < 0.01), 52.11 ± 0.56 % (P < 0.01), 53.42 ± 0.46 % (P < 0.01), and 46.70 ± 0.37% (P < 0.05), respectively. In addition, we show that SHJKS suppressed nuclear factor (NF)-κB activation induced by LPS plus PHA, leading to suppression of IκB-α phosphorylation and degradation. These results suggest that SHJKS might have regulatory effects on LPS plus PHA-induced cytokine production and NF-κB activation, which might explain its beneficial effect in the treatment of CI.     

ESTROGEN REGULATES CYTOKINE RELEASE IN HUMAN MAST CELLS

Estrogens are important for bone homeostasis and are classified as anti-resorptive agents. In ovariectomized rats, mast cell changes occurred during the activation of resorption. In addition, quantitative changes occurred in mast cell population residing near the site undergoing resorption. Considering these studies, mast cells may play a role in osteoporosis. Therefore, it is of paramount importance to study mast cell cytokine production also in the presence or absence of estrogen. When cultured in the absence of estrogen, human mast cells treated with PMA or A23187 demonstrated significantly greater release of TNF-α and IL-6 than cells grown under estrogen-depleted condition. Our results show that treatment of mast cells with estrogen prevented PMA or A23187-stimulated TNF-α or IL-6 release. These data provide evidence for a potent inhibition of cytokines by estrogen in human mast cells. This study may help to explain the association between mast cells and osteoporosis.     

Anti-inflammatory effects of isoacteoside from Abeliophyllum distichum

Abstract

Isoacteoside, a dihydroxypheynylethyl glycoside, is a major bioactive component of Abeliophyllum distichum (White Forsythia) which is a deciduous shrub native to the south and central areas of Korea. The present study is designed to evaluate the anti-inflammatory activities and underlying mechanisms of isoacteoside in human mast cell line, HMC-1 cells. We isolated isoacteoside from A. distichum. The anti-inflammatory effect of isoacteoside was investigated in HMC-1 cells by studying the following markers: phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) secretion and mRNA expression by ELISA and RT-PCR, respectively. In addition, mechanism related to anti-inflammatory was investigated by Western blotting. Isoacteoside significantly suppressed the production and mRNA expression of proinflammatory cytokines including IL-1β, IL-6, IL-8 and TNF-α in PMACI-stimulated HMC-1 cells without cytotoxicity. It was found that anti-inflammatory effects of isoacteoside are mediated by action on caspase-1, mitogen-activated protein kinases (c-Jun N-terminal kinase, p38, extracellular signal-regulated protein kinase) and nuclear factor-kappa B pathways. Taken together, the present findings provide new insights that isoacteoside may be a promising anti-inflammatory agent for inflammatory disorders.     

Anti-allergic effects of sinomenine by inhibition of prostaglandin D2 and leukotriene C4 in mouse bone marrow-derived mast cells

Sinomenine is an alkaloid compound and a prominent anti-allergic agent found in the root of the climbing plant Sinomenium acutum. However, its effects on the bone marrow-derived mast cell (BMMC) mediated allergy and inflammation mechanism remain unknown. In this study, the biological effects of sinomenine were evaluated while focusing on its effects on the allergic mediator in PMA plus A23187-stimulated BMMCs. An investigation was also conducted to determine its effects on the production of several allergic mediators including interleukin-6 (IL-6), prostaglandin D₂ (PGD₂), leukotriene C₄ (LTC₄), β-Hexosaminidase (β-Hex), and cyclooxygenase-2 (COX-2) protein. The results revealed that sinomenine inhibited the PMA plus A23187-induced production of IL-6, PGD₂, LTC₄, β-Hex, and COX-2 protein. Taken together, these findings indicate that sinomenine has the potential for use in the treatment of allergy.     

Effect of protein kinase inhibitors on IL-8/NAP-1 release from human umbilical vein endothelial cells

Several protein kinase inhibitors (PKIs) were investigated for their effects on IL-1β, TNFα and PMA-induced IL-8 production from human umbilical vein endothelial cells (HUVEC). IL-1β (ED₅₀ 0.07 ng/ml), TNFα (ED₅₀ 100 ng/ml) and PMA (ED₅₀ 20 ng/ml) induced IL-8 production that could be detected as early as 2 h following stimulation. Staurosporine, a potent but non-specific inhibitor of protein kinases, inhibited PMA-induced (IC₅₀ 2 nM) but not IL-1β or TNFα (IC50>200 nM) induced IL-8 production. Neither the cAMP-dependent PKI, KT5720, nor the tyrosine PKIs, genistein, tyrphostin (1–100 μM) or lavendustin A (0.0001–1 μM), inhibited IL-8 production elicited by IL-1β. However, the macrolide protein kinase inhibitor geldanamycin (IC₅₀=30 nM), but not the closely related analog herbimycin A (5–500 nM), inhibited IL-8 production by 60%. Northern blot analysis of IL-8 mRNA revealed that staurosporin suppressed mRNA increase following stimulation by PMA but not by IL-1. It is proposed that a novel protein kinase susceptible to geldanamycin inhibition may be involved in IL-1-mediated signal transduction.

     

Epigallocatechin-3-gallate inhibits secretion of TNF-alpha, IL-6 and IL-8 through the attenuation of ERK and NF-kappaB in HMC-1 cells

BACKGROUND: Epigallocatechin-3-gallate (EGCG) is a major form of tea catechin and has a variety of biological activities. In the present study, we investigated the effect of EGCG on the secretion of TNF-alpha, IL-6 and IL-8, as well as its possible mechanism of action by using the human mast cell line (HMC-1). METHODS: EGCG was treated before the activation of HMC-1 cells with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (A23187). To investigate the effect of EGCG on PMA+A23187-stimulated HMC-1 cells, ELISA, Western blot analysis, electrophorectic mobility shift assay and luciferase assay were used in this study. RESULTS: EGCG (100 microM) inhibited PMA+A23187-induced TNF-alpha, IL-6 and IL-8 expression and production. EGCG inhibited the intracellular Ca(2+) level. EGCG attenuated PMA+A23187-induced NF-kappaB and extracellular signal-regulated kinase (ERK1/2) activation, but not that of c-Jun N-terminal kinase or p38 mitogen-activated protein kinase. CONCLUSION: EGCG inhibited the production of TNF-alpha, IL-6 and IL-8 through the inhibition of the intracellular Ca(2+) level, and of ERK1/2 and NF-kappaB activation. These results indicate that EGCG may be helpful in regulating mast-cell-mediated allergic inflammatory response.     

[6]-Shogaol inhibits the production of proinflammatory cytokines via regulation of NF-κB and phosphorylation of JNK in HMC-1 cells

Abstract

[6]-Shogaol is a major bioactive component of Zingiber officinale. Although [6]-shogaol has a number of pharmacological activities including antipyretic, analgesic, antitussive and anti-inflammatory effects, the specific mechanisms of its anti-allergic effects have not been studied. In this study, we present the effects of [6]-shogaol on mast cell-mediated allergic reactions in vivo and in vitro. Sprague–Dawley rats received intradermal injections of anti-DNP IgE was injected into dorsal skin sites. After 48?h, [6]-shogaol was administered orally 1?h prior to challenge with DNP-HSA in saline containing 4% Evans blue through the dorsal vein of the penis. In addition, rat peritoneal mast cells (RPMCs) were cultured and purified to investigate histamine release. In vitro, we evaluated the regulatory effects of [6]-shogaol on the level of inflammatory mediators in phorbol 12-myristate 13-acetate plus calcium ionomycin A23187-stimulated human mast cells (HMC-1). [6]-Shogaol reduced the passive cutaneous anaphylaxis reaction compared to the control group, and histamine release decreased significantly following the treatment of RPMCs with [6]-shogaol. In HMC-1 cells, [6]-shogaol inhibited the production of TNF-α, IL-6 and IL-8, as well as the activation of nuclear factor-κB (NF-κB) and phosphorylation of JNK in compound 48/80-induced HMC-1 cells. [6]-shogaol inhibited mast cell-mediated allergic reactions by inhibiting the release of histamine and the production of proinflammatory cytokines with the involvement of regulation of NF-κB and phosphorylation of JNK.     

Gomisin N has anti-allergic effect and inhibits inflammatory cytokine expression in mouse bone marrow-derived mast cells

Gomisin N is a bioactive compound and a prominent anti-allergic agent found in the fruits of tree Schizandra chinensis. However, its effects on the bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanism remain unknown. In this study, the biological effects of gomisin were evaluated while focusing on its effects on the allergic mediator in PMA + A23187-stimulated BMMCs. The anti-allergic effect of gomisin has shown that inhibited PMA + A23187-induced interleukin-6 (IL-6) production. An investigation was also conducted to determine its effects on the production of several allergic mediators including prostaglandin D₂ (PGD₂), leukotriene C₄ (LTC₄), β-hexosaminidase (β-Hex), and cyclooxygenase-2 (COX-2) protein. The results revealed that gomisin inhibited the PMA + A23187-induced production of IL-6, PGD₂, LTC₄, β-Hex, and COX-2 protein. Taken together, these findings indicate that gomisin N has the potential for use in the treatment of allergy.     

Quantitative intracellular cytokine measurement: age-related changes in proinflammatory cytokine production

The proinflammatory cytokines play a central role in mediating cellular and physiological responses, and levels may reflect immune system effectiveness. In this study, the effect of ageing on the inflammatory response was examined using a novel method to detect production of the proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-α), IL-6 and IL-1β. Peripheral blood mononuclear cells (PBMC) obtained from healthy donors of different ages were incubated for 0, 24, 48 and 72 h with or without phorbol 12-myristate 13-acetate (PMA) stimulation. At each time point these cells were permeabilized and incubated with secondary conjugated FITC MoAbs specific for each cytokine. A flow cytometric system was developed to quantify specific intracellular fluorescence in T cells (CD3⁺) and monocytes (CD14⁺). TNF-α, IL-6 and IL-1β production in cell culture supernatants was also measured using ELISAs. In older subjects, flow cytometry detected significant increases in intracellular T cell TNF-α and IL-6 (P < 0.05). IL-1β was not detected in any of the T cell samples. Likewise, the monocytes of older subjects demonstrated increased intracellular levels of all three cytokines, but these increases were not significant (P > 0.05). These changes in intracellular proinflammatory cytokine levels may explain some of the exaggerated inflammatory responses seen in elderly patients.     

Anti-inflammatory activity of Korean folk medicine 'Sabaeksan'

Sabaeksan has been used for prevention and treatment of bronchial asthma and allergic asthma in Korea. To investigate the biological effect of Sabaeksan, we examined the effect of Sabaeksan on the phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-induced pro-inflammatory cytokines secretion in human mast cell line HMC-1 cells. Sabaeksan by itself had no effect on viability of HMC-1 cells. The effects of Sabaeksan on the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 from HMC-1 were evaluated with enzyme-linked immunosorbent assay. Sabaeksan (1 mg/ml) inhibited PMA plus A23187-induced TNF-alpha, IL-6, and IL-8 secretion by 43.86+/-5.26%, 56.39+/-3.65%, and 63.48+/-2.54%, respectively. Sabaeksan also inhibited the nuclear factor-kappa B (NF-kappaB) activation and IkappaBalpha degradation. Taken together, these results suggest that Sabaeksan inhibits the secretion of pro-inflammatory cytokines in HMC-1 cells through blockade of NF-kappaB activation.     

Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-μ antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-α (anti-TNF-α) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-α, but not other cytokines including IL-1β, IL-3, IL-5, IL-6, interferon-α (IFN-α) or IFN-γ, inhibited IL-4-mediated growth, and inhibition by TNF-α was blocked by anti-TNF-α Ab but not by control IgG. IL-4 had no effect on TNF-α binding by B cells while it decreased TNF-α production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-α by B cells, however, it enhanced TNF-α production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-α production.     

Differential effects of gangliosides on human IgE and IgG4 production

The effects of gangliosides on human IgE and IgG4 production were studied. Of the various gangliosides tested, only GM2 and GM3 inhibited the IgE and IgG4 production induced by interleukin (IL)-4 plus hydrocortisone (HC), or that induced by IL-13 plus HC, in human surface IgE- and IgG4-negative (sIgE^?, sIgG4^?) B cells without affecting the production of IgG1, IgG2, IgG3, IgM, IgA1 or IgA2. In contrast, GM1, GD1a, GD1b, GD3, GT1b and GQ1b were without effects. The GM2- and GM3-mediated inhibition was specific, since each was blocked by a corresponding antibody. Of the various factors tested, IL-6, IL-10, and tumor necrosis factor (TNF)-α enhanced the IgE and IgG4 production induced by IL-4 plus HC or by IL-13 plus HC, while IL-8 and transforming growth factor (TGF)-β inhibited these responses. However, only TNF-α counteracted the GM2- and GM3-mediated inhibition of IgE and IgG4 production, while IL-6, IL-10, anti-IL-8 monoclonal antibody and anti-TGF-β antibody failed to do so. Anti-TNF-α monoclonal antibody, but not control IgG1, not only inhibited IgE and IgG4 production in the absence of TNF-α but also blocked the counteraction of inhibition by TNF-α. In cultures containing IL-4 plus HC or IL-13 plus HC. GM2 and GM3 specifically inhibited TNF-α production without affecting TNF-α receptors, IL-6 production or IL-6 receptors. These results indicate that GM2 and GM3 inhibit IgE and IgG4 production by inhibiting endogenous TNF-α production.     

Inhibition of pro-inflammatory cytokines and inducible nitric oxide by extract of Emilia sonchifolia L. aerial parts

Emilia sonchifolia L. (Asteraceae) is used in ethnomedicine for the treatment of a wide array of inflammatory disorders. This practice has also been supported by scientific reports which showed that extracts of E. sonchifolia possess anti-inflammatory effects in rodents. However, the mechanism(s) through which the extracts produce these effects is not known. In this study, the effect of a methanol/methylene chloride extract of E. sonchifolia (ES) on the levels of IL-1β and TNF-α after an intraperitoneal lipopolysaccharide (LPS; 1?mg/kg) challenge was investigated in mice. The effect of ES on TNF-α and inducible nitric oxide (iNO) production by LPS-stimulated bone marrow-derived macrophages (BMMDM) was also investigated in vitro. BMMDM were pre-incubated for 2?h with ES (20, and 100 μg/mL) or with Pyrrolidine dithiocarbamate, PDTC (100 µM) and then activated with LPS, and then the IL-1β, TNF-α and NO production measured in the cell-free conditioned culture supernatant after 24?h of incubation. In groups of mice pre-treated with ES, the systemic levels of IL-1β and TNF-α induced by LPS were found to be significantly (p < 0.05) lower. In vitro, ES treatment caused a concentration-dependent decrease in LPS-inducible IL-1β, TNF-α, and NO production by BMDM compared to the effects of treatment of the cells with LPS alone without affecting the viability of the cells. The results of these studies suggest that treatment with ES alleviated inflammatory responses possibly through a suppression of pro-inflammatory mediators and cytokines such as IL-1β, TNF-α, and iNO.