Emerging evidence indicates that aldosterone and mineralocorticoid receptors (MRs) are associated with the pathogenesis of erectile dysfunction. However, the molecular mechanisms remain largely unknown. In this study, freshly isolated penile corpus cavernosum tissue from rats was treated with aldosterone, with or without MRs inhibitors. Nuclear factor (NF)-kappa B (NF-κB) activity was evaluated by real-time quantitative PCR, luciferase assay, and immunoblot. The results demonstrated that mRNA levels of the NF-κB target genes, including inhibitor of NF-κB alpha (IκB-α), NF-κB1, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), were higher after aldosterone treatment. Accordingly, phosphorylation of p65/RelA, IκB-α, and inhibitor of NF-κB kinase-β was markedly increased by aldosterone. Furthermore, knockdown of MRs prevented activation of the NF-κB canonical pathway by aldosterone. Consistent with this finding, ectopic overexpression of MRs enhanced the transcriptional activation of NF-κB by aldosterone. More importantly, the MRs antagonist, spironolactone blocked aldosterone-mediated activation of the canonical NF-κB pathway. In conclusion, aldosterone has an inflammatory effect in the corpus cavernosum penis, inducing NF-κB activation via an MRs-dependent pathway, which may be prevented by selective MRs antagonists. These data reveal the possible role of aldosterone in erectile dysfunction as well as its potential as a novel pharmacologic target for treatment.
Aldosterone/pharmacology* ; Animals ; Cytokines/biosynthesis* ; Gene Knockdown Techniques ; I-kappa B Kinase/antagonists & inhibitors* ; Interleukin-6/genetics* ; Male ; Mineralocorticoid Receptor Antagonists/pharmacology* ; NF-kappa B/genetics* ; Penis/metabolism* ; Protein Serine-Threonine Kinases/antagonists & inhibitors* ; RNA, Messenger/biosynthesis* ; Rats ; Rats, Inbred WKY ; Receptors, Mineralocorticoid/genetics* ; Signal Transduction/drug effects* ; Spironolactone/pharmacology* ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/biosynthesis* ; NF-kappaB-Inducing Kinase

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

Endotoxic responses to bacterial lipopolysaccharide (LPS) are triggered by Toll-like receptor 4 (TLR4) and involve the production of inflammatory mediators, including interleukin-6 (IL-6), by macrophages. The detailed mechanism of IL-6 production by macrophages in response to LPS has remained unclear, however. We now show that LPS induces IL-6 synthesis in mouse peritoneal macrophages via the leukotriene B4 receptor BLT2. Our results suggest that TLR4-MyD88 signaling functions upstream of BLT2 and that the generation of reactive oxygen species (ROS) by NADPH oxidase 1 (Nox1) and consequent activation of the transcription factor nuclear factor (NF)-kappaB function downstream of BLT2 in this response. These results suggest that a TLR4-MyD88-BLT2-Nox1-ROS-NF-kappaB pathway contributes to the synthesis of IL-6 in LPS-stimulated mouse macrophages.
Animals ; Cell Line ; Interleukin-6/*biosynthesis ; Leukotriene B4/metabolism ; Ligands ; Lipopolysaccharides/immunology ; Macrophages/immunology/*metabolism ; Macrophages, Peritoneal/immunology/metabolism ; Mice ; Myeloid Differentiation Factor 88/*metabolism ; NADH, NADPH Oxidoreductases/metabolism ; NF-kappa B/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*metabolism ; Signal Transduction

A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.
Animals ; Cell Cycle ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Fungal Polysaccharides ; pharmacology ; Immunity ; drug effects ; Interferon-gamma ; biosynthesis ; metabolism ; Interleukin-6 ; biosynthesis ; metabolism ; Macrophages ; immunology ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Pleurotus ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; biosynthesis ; metabolism ; Up-Regulation

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.
Antibodies ; administration & dosage ; immunology ; Bone Cements ; Fibroblasts ; immunology ; Gene Expression ; drug effects ; Humans ; Osteoprotegerin ; biosynthesis ; genetics ; Polymethyl Methacrylate ; administration & dosage ; Prostheses and Implants ; RANK Ligand ; biosynthesis ; genetics ; metabolism ; Receptors, Interleukin-6 ; immunology ; metabolism ; Synovial Fluid ; immunology ; metabolism

Ebosin is a novel exopolysaccharide produced by Streptomyces sp.139 with remarkable activity against rheumatic arthritis in vivo. In this paper, we reported effects of Ebosin on the inflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in THP-1 cells. With the special fluorogenic peptide as substrates, the enzymatic activities of interleukin-1beta converting enzyme (ICE) and TNFalpha-converting enzyme (TACE) were inhibited by Ebosin separately. Using the real-time reverse transcription polymerase chain reaction (real-time PCR), the mRNA synthesis of the three cytokines were identified decline separately by Ebosin. The secretion quantum of three cytokines in THP-1 cells with Ebosin was lower than that of normal THP-1 cells determined by ELISA assay and Western blotting. All of these results showed that Ebosin has remarkably suppressed synthesis of the three cytokines in THP-1 cells through different pathways. The primary study of Ebosin on anti-inflammation mechanism was promoted developing the new drugs treating rheumatic arthritis.
ADAM Proteins ; metabolism ; ADAM17 Protein ; Anti-Inflammatory Agents ; pharmacology ; Caspase 1 ; metabolism ; Cell Line, Tumor ; Humans ; Interleukin-1beta ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Polysaccharides, Bacterial ; biosynthesis ; pharmacology ; RNA, Messenger ; metabolism ; Streptomyces ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

T-helper (Th)17 cell responses are important for the development of neutrophilic inflammatory disease. Recently, we found that acetyl salicylic acid (ASA) inhibited Th17 airway inflammation in an asthma mouse model induced by sensitization with lipopolysaccharide (LPS)-containing allergens. To investigate the mechanism(s) of the inhibitory effect of ASA on the development of Th17 airway inflammation, a neutrophilic asthma mouse model was generated by intranasal sensitization with LPS plus ovalbumin (OVA) and then challenged with OVA alone. Immunologic parameters and airway inflammation were evaluated 6 and 48 h after the last OVA challenge. ASA inhibited the production of interleukin (IL)-17 from lung T cells as well as in vitro Th17 polarization induced by IL-6. Additionally, ASA, but not salicylic acid, suppressed Th17 airway inflammation, which was associated with decreased expression of acetyl-STAT3 (downstream signaling of IL-6) in the lung. Moreover, the production of IL-6 from inflammatory cells, induced by IL-17, was abolished by treatment with ASA, whereas that induced by LPS was not. Altogether, ASA, likely via its acetyl moiety, inhibits Th17 airway inflammation by blockade of IL-6 and IL-17 positive feedback.
Animals ; Aspirin/pharmacology/*therapeutic use ; Cell Polarity/drug effects/immunology ; Feedback, Physiological/*drug effects ; Interferon-gamma/deficiency/metabolism ; Interleukin-17/*metabolism/pharmacology ; Interleukin-6/biosynthesis/*metabolism ; Lipopolysaccharides/pharmacology ; Lung/drug effects/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Pneumonia/*drug therapy/*immunology/pathology ; Th17 Cells/drug effects/*immunology/pathology ; Transforming Growth Factor beta1/pharmacology

OBJECTIVECheongseoikki-tang (CIT, Korean), also called Qingshu Yiqi decoction () and Seisho-ekki-to (Japanese), is well known as an effective traditional combination of herbs for treating cardiovascular diseases. This study was to research its effects on bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanisms.

METHODSIn this study, the biological effect of Cheongseoikki-tang ethanol extract (CITE) was evaluated, focusing on its effects on the production of allergic mediators by phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187 (A23187)-stimulated BMMCs. These allergic mediators included interleukin-6 (IL-6), prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and β-hexosaminidase (β-hex).

RESULTSOur data revealed that CITE inhibited the production of IL-6, PGD2, LTC4, and β-hex induced by PMA plus A23187 (P<0.05).

CONCLUSIONThese findings indicate that CITE has the potential for use in the treatment of allergy.

Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Bone Marrow Cells ; pathology ; Calcimycin ; pharmacology ; Cell Degranulation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypersensitivity ; drug therapy ; pathology ; Interleukin-6 ; secretion ; Leukotriene C4 ; pharmacology ; Male ; Mast Cells ; drug effects ; pathology ; physiology ; Mice ; Mice, Inbred BALB C ; Prostaglandin D2 ; biosynthesis ; Tetradecanoylphorbol Acetate ; pharmacology ; beta-N-Acetylhexosaminidases ; metabolism

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.
Animals ; *Gene Expression Regulation/drug effects ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Male ; Mannitol/pharmacology ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Reactive Oxygen Species/*metabolism ; Rotenone/pharmacology ; Sodium Chloride/pharmacology ; Toll-Like Receptors ; Transcription Factors/genetics/*metabolism

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.
ATP-Binding Cassette Transporters/*metabolism ; Adult ; Aged ; Arthritis, Rheumatoid/*pathology ; CD4-Positive T-Lymphocytes/metabolism ; Calgranulin B/metabolism ; Cell Differentiation/*immunology ; Fibroblasts/*metabolism/pathology ; Humans ; Inflammation Mediators/metabolism ; Interleukin-17/metabolism ; Interleukin-6/*biosynthesis ; Middle Aged ; Signal Transduction/immunology ; Synovial Fluid/cytology ; Synovial Membrane/metabolism/pathology ; Th17 Cells/*pathology ; Toll-Like Receptor 4/metabolism ; *Up-Regulation