IOX1 activity as sepsis therapy and an antibiotic against multidrug-resistant bacteria.

Sepsis is caused by organ dysfunction initiated by an unrestrained host immune response to infection. The emergence of antibiotic-resistant bacteria has rapidly increased in the last decades and has stimulated a firm research platform to combat infections caused by antibiotic-resistant bacteria that cannot be eradicated with conventional antibiotics. Strategies like epigenetic regulators such as lysine demethylase (Kdm) has received attention as a new target. Thus, we sought to investigate the epigenetic mechanisms in sepsis pathophysiology with the aim of discovering new concepts for treatment. A transcriptome analysis of dendritic cells during their inflammatory state identified Kdm as a critical molecule in sepsis regulation. Next, 8-hydroxyquinoline-5-carboxylic acid (IOX1) ability to control endotoxemia induced by Lipopolysaccharide and bacterial sepsis was demonstrated. IOX1 has been shown to regulate endotoxemia and sepsis caused by Escherichia coli and carbapenem-resistant Acinetobacter baumannii and has also contributed to the suppression of multidrug-resistant bacterial growth through the inhibition of DNA Gyrase. These findings show that IOX1 could be a component agent against bacterial sepsis by functioning as a broad-spectrum antibiotic with dual effects.

Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity.

BACKGROUND: Complementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo. METHODS: The effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis. RESULTS: LERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells. CONCLUSION: This study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.

Ellagic acid modulates LPS-induced maturation of dendritic cells through the regulation of JNK activity.

Ellagic acid (EA) is a well- known phytochemical that modulates various cellular processes. It is present in a variety of foods, including grapes, strawberries, and nuts. However, the influence of EA on immunological responses is not well defined. Here, we investigated the effects of EA on the lipopolysaccharide (LPS)-induced bone marrow-derived dendritic cells (BMDCs). EA was not cytotoxic to DCs. EA suppressed LPS-induced expression of costimulatory molecules (CD80 and CD86), but it did not suppress the expression of major histocompatibility complex (MHC) class I and MHC class II in BMDCs. In particular, EA blocked LPS-induced c-Jun N-terminal kinase (JNK) activation. LPS-mediated expression of proinflammatory cytokines (IL-12 and IFN-γ) was diminished by EA. We also confirmed that levels of IL-12 and IFN-γ were not influenced by EA in the presence of a JNK inhibitor. Taken together, these data demonstrate that EA regulates the immune response through the modulation of LPS-induced DC maturation.

Rhamnogalacturonan II is a Toll-like receptor 4 agonist that inhibits tumor growth by activating dendritic cell-mediated CD8+ T cells.

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1ß, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.

Interplay of reactive oxygen species, intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin.

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents of natural product origin. We previously reported anti-proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC-3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase-3-dependent pathway that is activated due to dysregulated mitochondrial function. DPT-treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca (i)(2+) surge, increased mitochondrial membrane potential (MMP, ΔΨ(m)), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase-3 activation, which in turn induced apoptosis. The antioxidant N-acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca (i)(2+) surge, on the other hand the Ca(2+) chelator BAPTA inhibited the Ca( i)(2+) overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca(2+) flux. This suggested that both ROS and Ca( i)(2+) signaling play roles in the increased MMP via Ca (i)(2+)-dependent and/or -independent mechanisms, since ΔΨ(m) elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS- and Ca( i)(2+)-activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca(2+) flux homeostasis.

Sulforaphane inhibits the Th2 immune response in ovalbumin-induced asthma.

Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane), belonging to a family of natural compounds that are abundant in broccoli, has received significant therapeutic interest in recent years. However, the molecular basis of its effects remains to be elucidated. In this study, we attempt to determine whether sulforaphane regulates the inflammatory response in an ovalbumin (OVA)-induced murine asthma model. Mice were sensitized with OVA, treated with sulforaphane, and then challenged with OVA. Sulforaphane administration significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Additionally, sulforaphane suppressed the increase in the levels of SOCS-3 and GATA-3 and IL-4 expression in the OVA-challenged mice. Collectively, our results demonstrate that sulforaphane regulates Th2 immune responses. This sutdy provides novel insights into the regulatory role of sulforaphane in allergen-induced Th2 inflammation and airway responses, which indicates its therapeutic potential for asthma and other allergic diseases.

RG-II from Panax ginseng C.A. Meyer suppresses asthmatic reaction.

In asthma, T helper 2 (T(H)2)-type cytokines such as interleukin (IL)-4, IL-5, and IL-13 are produced by activated CD4(+) T cells. Dendritic cells played an important role in determining the fate of naive T cells into either T(H)1 or T(H)2 cells. We determined whether RG-II regulates the T(H)1/T(H)2 immune response by using an ovalbumin-induced murine model of asthma. RG-II reduced IL-4 production but increased interferon- gamma production, and inhibited GATA-3 gene expression. RG-II also inhibited asthmatic reactions including an increase in the number of eosinophils in bronchoalveolar lavage fluid, an increase in inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyperresponsiveness. This study provides evidence that RG-II plays a critical role in ameliorating the pathogenic process of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of RG-II in terms of its effects in a murine model of asthma.

Quercetin regulates Th1/Th2 balance in a murine model of asthma.

Quercetin is found to be the most active of the flavonoids in studies and many medicinal plants owe much of their activity to their high Quercetin content. Quercetin has demonstrated significant anti-inflammatory activity because of direct inhibition of several initial processes of inflammation. However, its anti-allergic effect in the Th1/Th2 immune response was poorly understood. Recently, it was shown that T-bet and GATA-3 were master Th1 and Th2 regulatory transcription factors. In this study, we have attempted to determine whether Quercetin regulates Th1/Th2 cytokine production, T-bet and GATA-3 gene expression in OVA-induced asthma model mice. Quercetin reduced the increased levels of IL-4, Th2 cytokine production in OVA-sensitized and -challenged mice. The other side, it increased IFN-gamma, Th1 cytokine production in Quercetin administrated mice. We also examined to ascertain whether Quercetin could influence Eosinophil peroxidase (EPO) activity. The administration of Quercetin before the last airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. Accordingly, this study may provide evidence that Quercetin plays a critical role in the amelioration of the pathogenetic process of asthma in mice. These findings provide new insight into the immunopharmacological role of Quercetin in terms of its effects in a murine model of asthma, and also broaden current perspectives in our understanding of the immunopharmacological functions of Quercetin.

Biotransformation of Korean Panax ginseng by Pectinex.

Ginsenosides comprise the major component of ginseng exhibit various types of biological activity, including antiinflammatory and antitumor effects. In these pharmacological actions, it is thought that these activities are carried out by the metabolites of ginsenosides metabolized by human intestinal microflora. It has also been reported that their clinical efficacy varies with the hydrolyzing potential of the components of the intestinal microflora. We tried to develop a process for metabolizing ginsenosides to compound K using food-grade enzymes, which can be used commercially. Among these, Pectinex proved to be the most effective mediator of the catabolism of ginsenosides to compound K. The optimal conditions for this biotransformation were determined to be as follows: 10 to 15% rootlet ginseng, pH 5, 50 degrees C, and 2 to 3 d of incubation, to yield 20.0 mg of compound K/g of rootlet ginseng. We suggest that the metabolism of ginseng to compound K in the presence of Pectinex has many advantages over previous methods, in respects of use of raw, non-extracted rootlet ginseng, which do not require more organic solvents and evaporation apparatus. Potential metabolites PG1, PG2, PG3, and PG4 were detected in Pectinex-treated rootlet ginseng using by TLC and HPLC and, among them, PG4 was identified as compound K by TLC, HPLC, and MS. Additional studies will be carried out to determine the structure of these metabolites of ginseng and to understand the relationship between their structures and activities.

Beta-lapachone, a quinone isolated from Tabebuia avellanedae, induces apoptosis in HepG2 hepatoma cell line through induction of Bax and activation of caspase.

The DNA topoisomerase inhibitor beta-lapachone is a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae) in South America. It has been reported to possess a wide range of pharmacological properties, and is a promising cancer chemopreventive agent. In this study, the effects of beta-lapachone on the growth of the human hepatoma cell line HepG2 were investigated. The results showed that beta-lapachone inhibits the viability of HepG2 by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. Reverse transcription-polymerase chain reaction and immunoblotting results indicated that treatments of cells with beta-lapachone resulted in down-regulation of anti-apoptotic Bcl-2 and Bcl-X(L) and up-regulation of pro-apoptotic Bax expression. beta-Lapachone-induced apoptosis was associated with a proteolytic activation of caspase-3 and -9 and degradation of poly(ADP-ribose) polymerase protein. However, beta-lapachone treatment did not affect the inhibitor of apoptosis proteins family and the Fas/FasL system. Taken together, our study indicated that beta-lapachone may have potential as a chemopreventive agent for liver cancer.

Epigallocatechin-3-gallate protects toluene diisocyanate-induced airway inflammation in a murine model of asthma.

Epigallocatechin-3-gallate (EGCG), a major form of tea catechin, has anti-allergic properties. To elucidate the anti-allergic mechanisms of EGCG, we investigated its regulation of matrix metalloproteinase (MMP-9) expression in toluene diisocyanate (TDI)-inhalation lung tissues as well as TNF-alpha and Th2 cytokine (IL-5) production in BAL fluid. Compared with untreated asthmatic mice those administrated with EGCG had significantly reduced asthmatic reaction. Also, increased reactive oxygen species (ROS) generation by TDI inhalation was diminished by administration of EGCG in BAL fluid. These results suggest that EGCG regulates inflammatory cell migration possibly by suppressing MMP-9 production and ROS generation, and indicate that EGCG may be useful as an adjuvant therapy for bronchial asthma.

Water extract of Cordyceps militaris enhances maturation of murine bone marrow-derived dendritic cells in vitro.

Water extract (WE) of Cordyceps militaris has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not been known. In this study, we investigated whether water extract of C. militaris induces the phenotypic and functional maturation of dendritic cells (DC). It profoundly increased CD40, CD54, CD80, CD86, and MHC class II expression in murine bone marrow (BM)-derived myeloid DC. Endocytosis was assessed by the uptake of FITC-dextran and FITC-albumin. The ability of unstimulated DC (UT-DC) to uptake dextran and albumin was higher than that of WE- or LPS-stimulated DC (LPS-DC). Also, UT-DC secreted a low concentration of IL-12, while WE- or LPS-DC secreted higher levels of IL-12 than UT-DC. WE not only formed morphologically mature DC and clusters, but also induced predominantly functional maturation. Moreover, WE is shown to promote the cytotoxicity of specific-cytotoxic T lymphocyte (CTL) induced by DC which were pulsed with P815 tumor-lysate during the stage of antigen presentation. These results suggest that DC maturation by WE can play a critical role in the improvement of the immunoregulatory function in patients with impaired host defense.

Growth inhibition of U937 leukemia cells by aqueous extract of Cordyceps militaris through induction of apoptosis.

Cordyceps militaris is a traditional herbal ingredient, which has been used for patients suffering from cancer in Oriental medicine. In the present study, we investigated the biochemical mechanisms of anti-proliferative effects by aqueous extract of C. militaris (AECM) in human leukemia U937 cells. It was found that AECM could inhibit cell growth of U937 cells in a dose-dependent manner, which was associated with morphological change and apoptotic cell death such as formation of apoptotic bodies and DNA fragmentation. We observed the down-regulation of anti-apoptotic Bcl-2 expression and proteolytic activation of caspase-3 in AECM-treated U937 cells. However, AECM did not affect the pro-apoptotic Bax expression and activity of caspase-9. Furthermore, Western blotting and RT-PCR revealed that AECM treatment caused a dose-dependent inhibition of cyclooxygenase-2 and prostaglandin E2 accumulation. Taken together, these results indicated that the anti-proliferative effects of AECM were associated with the induction of apoptotic cell death through regulation of several major growth regulatory gene products such as Bcl-2 family expression and caspase protease activity, and AECM may have therapeutic potential in human leukemia treatment.

Acidic polysaccharide isolated from Phellinus linteus enhances through the up-regulation of nitric oxide and tumor necrosis factor-alpha from peritoneal macrophages.

Medicinal mushrooms are increasingly used to treat a wide variety of disease processes. Aqueous extract from the fruiting body or mycelia of Phellinus linteus has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the mechanisms underlying its tumoricidal effects are poorly understood. The tumoricidal activity of peritoneal macrophages (PM) cultured with acidic polysaccharide (PL) isolated from Phellinus linteus against B16 melanoma cells was enhanced in a dose-dependent manner; growth inhibition increased 4-fold with 200 microg/ml of PL. To further characterize the mechanisms of PL, we investigated the effects of PL on phagocytosis and the release of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and reactive oxygen intermediates (ROI). To investigate the phagocytosis of PM, the uptake of Dextran (Dex)-FITC between PL-untreated and PL-treated PM was compared. We found some augment in phagocytosis of PL-treated PM compared untreated group. PL stimulated a dose-dependent increase in NO and TNF-alpha, but not in ROI production in PM. We suggested that PL has cytotoxicity against Yac-1 cells through the up-regulation of NO and TNF-alpha production. Also, PL enhanced the expression of costimulatory molecules, CD80 and CD86, and major histocompatibility complex (MHC) molecules II in PM. The ability of PL upon the up-regulation of these surface molecules involved in antigen-presenting processes may, by inference, activate T-cell-mediated immunity against malignant cells in vivo. Taken together, these results suggest that PL act as an effective immunomodulator and enhances the anti-tumoral activity of PM.

Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB.

The effects of epigallocatechin-3-gallate (EGCG) on dendritic cells (DC) maturation were investigated. EGCG, in a dose-dependent manner, profoundly inhibited CD80, CD86, and MHC class I and II expression on bone marrow-derived murine myeloid DC. EGCG restored the decreased dextran-FITC uptake and inhibited enhanced IL-12 production by LPS-treated DC. EGCG-treated DC were poor stimulators of nai;ve allogeneic T-cell proliferation and reduced levels of IL-2 production in responding T cells. EGCG-pretreated DC inhibited LPS-induced MAPKs, such as ERK1/2, p38, JNK, and NF-kappaB p65 translocation. Therefore, the molecular mechanisms by which EGCG antagonized LPS-induced DC maturation appeared to involve the inhibition of MAPK and NF-kappaB activation. These novel findings provide new insight into the immunopharmacological role of EGCG and suggest a novel approach to the manipulation of DC for therapeutic application of autoimmune and allergic diseases.

Alleviation of experimental septic shock in mice by acidic polysaccharide isolated from the medicinal mushroom Phellinus linteus.

This study reports that acidic polysaccharide (PL) isolated from Phellinus linteus alleviated the septic shock induced by high dose lipopolysaccharide (LPS) injection in mice. To examine the origin of this effect, we investigated cytokine production in serum and the expression of MHC II in B cells and macrophages in areas of inflammation. Pretreatment with PL 24 h before LPS administration resulted in a significant inhibition of up to 68% of circulating tumor necrosis factor (TNF)-alpha, a moderate reduction of 45% of interleukine (IL)-12 and 23% of IL-1beta, but no significant reduction in IL-6. In addition, the expression of MHC II in B cells and macrophages was examined. Our results show that LPS-stimulated cytokine release and the level of MHC II can be modulated by in vivo administration of soluble PL in mice. The decrease of IL-1beta, IL-12 and TNF-alpha in sera and the down-modulation of MHC II during septic shock may contribute to the long survival of mice by PL. Administration of PL in vivo decreases IL-2, IFN-gamma and TNF-alpha production in splencotyes and enhances spontaneous cell apoptosis in macrophages and lymphocytes stimulated with LPS in vitro. Thus, part of the anti-inflammatory effects of PL treatment in vivo may result from the enhanced apoptosis of a portion of the activated macrophages and lymphocytes. The ability of PL to significantly reduce the TNF-alpha production indicates the potential of the polysaccharides in possible therapeutic strategies that are based on down-regulation of TNF-alpha.

Acidic polysaccharide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C.

Mushroom polysaccharides are increasingly being utilized to treat a wide variety of diseases. Aqueous extracts from the Phellinus linteus have been reported to have anti-tumor and immunomodulatory properties. In particular, acidic polysaccharide (PL) isolated from P. linteus induced a secretory and cellular macrophage response. However, the exact mechanism by which PL regulates the macrophage functions remains unclear. PL-treated murine peritoneal macrophages in vitro and in vivo dramatically induced the production of NO. PL enhanced the lytic death of B16 cells through the production of NO. The present study examined signal molecules that may participate in PL-elicited responses by macrophages. The data demonstrated that a protein kinase C (PKC) inhibitor, staurosporine, and a protein tyrosine kinase (PTK) inhibitor, genistein, inhibited the tumoricidal activity of macrophages induced by PL. In addition, these inhibitors blocked the production of NO and the expression of surface molecules in PL-stimulated macrophages. Furthermore, CD11b/CD18 possibly mediates PL-induced cell activation. These results suggest that PL stimulates NO production for tumoricidal activity and induces cell-mediated immunity by increasing surface molecules, and the process may be a mechanism by which PL produces its therapeutic effects.

Proteoglycan isolated from Phellinus linteus activates murine B lymphocytes via protein kinase C and protein tyrosine kinase.

Medicinal mushrooms are increasingly used to treat a wide variety of disease processes. Aqueous extract from the fruiting body or mycelia of Phellinus linteus has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not been known. In the present study, we investigated whether proteoglycan (PL) isolated from P. linteus has an effect on the immunomodulatory activities of the murine splenic lymphocytes (MSLs). Treatment with PL caused a four-fold augmentation in [3H]thymidine incorporation compared to untreated control group in MSLs. Flow cytometric analysis indicated that the affected cell population was mainly CD19(+) cells, but not CD3(+) cells. These data suggested that the main target of PL was the B cells, but not T cells. PL also enhanced the expression of co-stimulatory molecules, CD80 and CD86, in murine B cells in a time-dependent manner. Accordingly, we investigated if intracellular [Ca(2+)] and reactive oxygen intermediates (ROI) were the principal downstream components that contributed to PL-induced activation, with respect to both increases of proliferation and induction of co-stimulatory molecules. However, PL has no influence on the [Ca(2+)] concentration and the ROI formation in murine B cells, whereas the genistein, protein tyrosine kinase (PTK) inhibitor or staurosporine, protein kinase C (PKC), blocked the proliferation and the induction of co-stimulatory molecules, CD80 and CD86, in B cells stimulated with PL. Taken together, these data suggest that PL is a biological response modifier that stimulates proliferation and expression of co-stimulatory molecules in B cells, probably by regulating PTK and PKC signaling pathways.

Oral administration of proteoglycan isolated from Phellinus linteus in the prevention and treatment of collagen-induced arthritis in mice.

To examine whether oral administration of proteoglycan derived from Phellinus linteus, which is known as the medicinal mushroom, can prevent or treat collagen-induced arthritis (CIA) in mice as experimental model of autoimmune disease. CIA was induced by intradermal injection of type II collagen (CII) emulsified with complete freund's adjuvant (CFA) into the base of the tail (on day 7) followed by a booster injection on day 21 into the footpad. To examine the ability of proteoglycan to effect the inhibition of CIA, doses of proteoglycan were orally administered on day 0 (pre-administration) or day 28 (post-administration) at two groups. The inhibition of CIA by oral administration of proteoglycan was associated with decrease in anti-CII IgG and IgG2a antibodies (Abs) as well as varying kinds of cytokines including IL-12, TNF-alpha, and IFN-gamma. The results showed that administration of proteoglycan was followed by decrease of CIA of the mice in pre- and post-administration groups. Our findings suggest that immunomodulating proteoglycan isolated from P. linteus may be crucially involved in the prevention and treatment of autoimmune joint inflammation such as rheumatoid arthritis, although no definite role of anti-CII Abs in the human disease has been established.