ABSTRACT
AIM OF THE STUDY: The roots of Dichroa febrifuga Lour. have been used as a traditional antimalarial drug and also used in the treatment of productive cough and unstable fever caused by infection in China and Korea. In this study, we evaluated the anti-inflammatory effect and underlying molecular mechanism of aqueous extract of Dichroa febrifuga (AEDF) in C57BL/6 mouse peritoneal macrophages. MATERIALS AND METHODS: The effect of AEDF on proinflammatory cytokine (IL-1beta and IL-6) production was analyzed by ELISA and real-time RT-PCR. The effects of AEDF on NF-kappaB/IkappaB-alpha/IKK were measured by reporter assay (in RAW 264.7 cells), EMSA, Western blotting and kinase assay. The effects of AEDF on Akt and MAPKs activity were assayed by Western blotting. RESULTS: AEDF inhibited the production of IL-1beta and IL-6, NF-kappaB activation, IkappaB-alpha degradation, and IKK, Akt, ERK1/2 and JNK activities in LPS-stimulated mouse peritoneal macrophages. CONCLUSIONS: These results suggest that AEDF inhibits proinflammatory cytokine (IL-1beta and IL-6) production in LPS-stimulated mouse peritoneal macrophages, and that these effects are mediated by the inhibition of the activity of IKK/IkappaB/NF-kappaB and the phosphorylation of Akt, ERK1/2, and JNK. Our results provide a molecular basis for understanding the inhibitory effects of Dichroa febrifuga roots on endotoxin-mediated inflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Hydrangeaceae , Interleukin-1beta/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Plant Extracts/pharmacology , Animals , I-kappa B Proteins/antagonists & inhibitors , Interleukin-1beta/biosynthesis , Interleukin-6/biosynthesis , Lipopolysaccharides , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NF-KappaB Inhibitor alpha , Phosphorylation , Plant Roots , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , RNA, Messenger/biosynthesis , RNA, Messenger/drug effectsABSTRACT
The thorns of Gleditsia sinensis LAM. (Leguminosae) have been used in traditional medicine for the treatment of inflammatory diseases including swelling, suppuration, carbuncle and skin diseases in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of Gleditsia sinensis thorns in RAW 264.7 macrophages. The aqueous extract of Gleditsia sinensis thorns (AEGS) inhibited LPS-induced NO secretion as well as inducible nitric oxide synthase (iNOS) expression, without affecting cell viability. Furthermore, AEGS suppressed LPS-induced NF-kappaB activation, phosphorylation and degradation of IkappaB-alpha, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). These results suggest that AEGS has the inhibitory effects on LPS-induced NO production and iNOS expression in macrophages through blockade in the phosphorylation of MAPKs, following IkappaB-alpha degradation and NF-kappaB activation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Gleditsia , Lipopolysaccharides/pharmacology , Macrophages/drug effects , NF-kappa B/antagonists & inhibitors , Nitric Oxide/antagonists & inhibitors , Plant Extracts/pharmacology , Animals , Cells, Cultured , I-kappa B Proteins/metabolism , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NF-KappaB Inhibitor alpha , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/geneticsABSTRACT
Bee venom (BV) has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in BV-induced apoptosis are still uncharacterized in human leukemic cells. In the present study, we report that BV induces apoptosis in leukemic U937 cells through downregulation of ERK and Akt signal pathway. Furthermore, BV-induced apoptosis was accompanied by downregulation of Bcl-2, activation of caspase-3 and a subsequent poly(ADP-ribose)polymerase (PARP) cleavages. The induction of apoptosis also was accompanied by the downregulation of the inhibitor of apoptosis protein (IAP) family proteins. Caspase-3 inhibitor, z-DEVD-fmk, was significantly capable of restoring cell viability and BV-induced apoptosis through caspase-3 activation was significantly attenuated in Bcl-2-overexpressing cells. These results indicate that downregulation of Bcl-2 plays a major role in the initiation as an activator of a caspase-3 involved with BV-induced apoptosis. BV also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. PD98059 (an inhibitor of ERK) or LY294002 (an inhibitor of Akt), but not an inhibitor of p38 MAPK and JNK, significantly decreased cell viability and increased lactate dehydrogenase (LDH) release. The results indicated that key regulators in BV-induced apoptosis are Bcl-2 and caspase-3 in human leukemic U937 cells through downregulation of the ERK and Akt signal pathway.
Subject(s)
Apoptosis/drug effects , Bee Venoms/pharmacology , Cell Proliferation/drug effects , Animals , Bone Marrow Cells , Caspase 3/metabolism , Cell Survival/drug effects , Collagen Type XI/metabolism , DNA Fragmentation , Down-Regulation , Extracellular Signal-Regulated MAP Kinases/metabolism , HL-60 Cells , Humans , K562 Cells , L-Lactate Dehydrogenase/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , U937 CellsABSTRACT
Seungma-galgeun-tang (SGT) has been used for treatment of chronic diseases in the folk medicine recipe. Since nitric oxide (NO) is one of the major inflammatory parameters, we first studied the effects of SGT on NO production in lipopolysaccharide (LPS)-stimulated BV-2 microglia. SGT inhibited the secretion of NO in BV-2 microglia, without affecting cell viability. The protein level of inducible nitric oxide synthase (iNOS) was decreased by SGT and SGT also inhibited production of PGE(2) and expression of Cox-2. Proinflammatory cytokines, such as TNF-alpha, IL-1beta and IL-12, were inhibited by SGT in a dose-dependent manner and SGT blocked the activation of NF-kappaB, which was considered to be a potential transcription factor for the expression of iNOS, COX-2 and proinflammatory cytokines. SGT also blocked the degradation of IkappaB and activation (decrease of cytosolic p65) of NF-kappaB, p65. These results suggest that SGT could exert its anti-inflammatory actions by suppressing the synthesis of NO through inhibition of NF-B activity.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Inflammation/prevention & control , Microglia/metabolism , NF-kappa B/metabolism , Animals , Base Sequence , Blotting, Western , Cell Line , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/drug effects , DNA Primers , Dinoprostone/antagonists & inhibitors , Down-Regulation/drug effects , Hydrolysis , Inflammation/metabolism , Inflammation Mediators/antagonists & inhibitors , Mice , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/biosynthesis , RNA, Messenger/geneticsABSTRACT
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.
Subject(s)
Bone Marrow Cells/cytology , Cordyceps/chemistry , Dendritic Cells/drug effects , Materia Medica/pharmacology , Animals , Antigens, CD/biosynthesis , Cell Survival/drug effects , Cells, Cultured , Cytokines/biosynthesis , Dendritic Cells/metabolism , Dendritic Cells/physiology , Endocytosis/drug effects , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads, and has been used as an Oriental drug. However, little is known about the effect of Chan Su on the growth of human cancer cells. This study was undertaken to investigate the underlying mechanism of Chan Su-induced apoptosis in a human bladder carcinoma cell line, T24. The effects of this compound were also tested on cyclooxygenase (COX) activity. Treatment of T24 cells with Chan Su resulted in the inhibition of viability and induction of apoptosis in a concentration-dependent manner, which was proved by trypan blue counts, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. Apoptosis of T24 cells by Chan Su was associated with a down-regulation of anti-apoptotic Bcl-2 and Bcl-X(S/L) expression and an up-regulation of pro-apoptotic Bax expression. Chan Su treatment induced the proteolytic activation of caspase-3 and caspase-9, and a concomitant degradation of poly(ADP-ribose)-polymerase and beta-catenin protein. Furthermore, Chan Su decreased the levels of COX-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with an inhibition in prostaglandin E(2) synthesis. Taken together, these findings partially provide novel insights into the possible molecular mechanisms of the anti-cancer activity of Chan Su.
Subject(s)
Apoptosis/drug effects , Bufanolides/pharmacology , Amphibian Venoms/pharmacology , Animals , Blotting, Western , Caspase 3 , Caspase 9 , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclooxygenase 1 , Cyclooxygenase 2 , Cytoskeletal Proteins/metabolism , Dinoprostone/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoenzyme Techniques , Membrane Proteins , Poly(ADP-ribose) Polymerases/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/metabolism , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , bcl-2-Associated X Protein , bcl-X Protein , beta CateninABSTRACT
Spirodela polyrhixa Schleid has been used in folk medicine to treat inflammatory diseases. Since nitric oxide (NO) is one of the major inflammatory parameters, we studied the effect of aqueous extracts of Spirodela polyrhixa (AESP) on NO production in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. AESP inhibited the secretion of NO in macrophages, without affecting cell viability. The protein level of inducible nitric oxide synthase (iNOS) in peritoneal macrophages was also decreased by AESP. Transient transfection assay of reporter plasmid and gel shift assay indicated that AESP blocked the activation of nuclear factor-kappa B (NF-kappa B), which was considered to be a potential transcription factor for iNOS expression. AESP also blocked the phosphorylation and degradation of inhibitory protein I kappa B-alpha (I kappa B-alpha). These results suggest that AESP could exert its anti-inflammatory actions by suppressing the synthesis of NO through inhibition of NF-kappa B activity.
Subject(s)
Alismatales , Anti-Inflammatory Agents/pharmacology , Macrophages/drug effects , NF-kappa B/antagonists & inhibitors , Nitric Oxide/biosynthesis , Phytotherapy , Plant Extracts/pharmacology , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Dose-Response Relationship, Drug , Lipopolysaccharides , Macrophages/enzymology , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/drug effects , Nitric Oxide Synthase Type II , Plant Extracts/administration & dosage , Plant Extracts/therapeutic useABSTRACT
AIM: To study the anti-inflammatory effects of aqueous extract from Dichroa febrifuga root (AEDF) for suppression in the process of lipopolysaccharide (LPS)-induced sepsis in the rat liver. METHODS: The inhibitory effect of AEDF on the alteration of inflammatory proteins was investigated by Western blot and immunohistochemical analysis. RESULTS: Western blot analysis showed that the level of nuclear factor (NF)-kappaBp65 was markedly up-regulated and (I)-kappaBalpha was down-regulated by LPS (8 mg/kg) challenge. However, AEDF 100 mg/kg inhibited induction of NF-kappaBp65 and degradation of I-kappaBalpha in the liver of LPS-challenged rats. Immunohistochemical analysis showed that while the expression of the NF-kappaBp65, tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS) tended to increase, that of I-kappaBalpha was decreased in the hepatocytes of rats challenged with LPS. A slight decline of NF-kappaBp65, TNF-alpha and iNOS, but an increase of I-kappaBalpha were observed in the hepatocytes of the rats pretreated with AEDF. CONCLUSION: AEDF may act as a therapeutic agent for inflammatory disease through a regulation of inflammation-related proteins.
