OBJECTIVEBuckwheat has been considered as a potential source of nutraceutical components on the world market of probiotic foodstuffs. The purpose of this study was to evaluate the effects of tartary buckwheat (Fagopyrum tataricum) sprouts on oxidation and pro-inflammatory mediators.

METHODSThe anti-oxidant effects of buckwheat extract (BWE) and rutin were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)- and nitric oxide (NO)-scavenging activities, serum peroxidation and chelating assays. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used to evaluate anti-inflammatory activities of buckwheat and rutin. NO production in LPS-stimulated RAW264.7 cells was determined by using Griess reagent. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) p65 subunit in cytosolic and nuclear portions were determined by Western blot analysis. Also, the production of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay.

RESULTSInhibitory concentration 50 values for DPPH- and NO-scavenging activities of BWE were 24.97 and 72.54 μg/mL respectively. BWE inhibited serum oxidation and possessed chelating activity. Furthermore, BWE inhibited IL-6 and TNF-α production in LPS-stimulated RAW264.7 cells. Also, BWE inhibited iNOS and COX-2 expression and NF-κB p65 translocation.

CONCLUSIONBuckwheat sprouts possessed strong antioxidant activity and inhibited production of pro-inflammatory mediators in the applied model systems. Thus, buckwheat can be suggested to be beneficial in inflammatory diseases by inhibiting the free radicals and inflammatory mediators.

Animals ; Cells, Cultured ; Cyclooxygenase 2 ; analysis ; Fagopyrum ; Free Radical Scavengers ; pharmacology ; Inflammation Mediators ; antagonists & inhibitors ; Interleukin-6 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; analysis ; Plant Extracts ; pharmacology ; Tumor Necrosis Factor-alpha ; biosynthesis

Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-gamma in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-beta, is important in wound healing. We investigated the role of FGF2 in IFN-gamma-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-gamma-induced emphysema, lung targeted IFN-gamma transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 microg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-gamma in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-gamma but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-gamma-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.
Animals ; Asthma/drug therapy/*prevention & control ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Emphysema/drug therapy/*prevention & control ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2/deficiency/*metabolism/*therapeutic use ; Flow Cytometry ; Inflammation/immunology ; Interferon-gamma/*biosynthesis/genetics ; Interleukin-13 ; Lipopolysaccharides/administration & dosage/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Pulmonary Eosinophilia ; Recombinant Proteins/administration & dosage/therapeutic use

OBJECTIVETo study the inhibitory effect of Fuzheng Yiliu Granule (FYG) on hepatocellular cancer (HCC) and investigate the mechanism mediating its bioactivity.

METHODSH22 tumor-bearing ICR mice were treated with FYG [3.6 g/(kg·d)] for 5 days. Tumor volume and tumor weight, percentages of CD3(+), CD4(+), CD8(+), and natural killer (NK) cells in peripheral blood, tumor apoptosis and serum levels of interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) were evaluated. FYG-containing serum was prepared from SD rats treated for 7 days [high dose 3.6 g/(kg·d); middle dose 1.8 g/(kg·d); low dose 0.9 g/(kg·d)]. Cell cycle, cell viability, and apoptosis were evaluated after HepG2 cell line was cultured in FYG-containing serum for 48 h. The levels of IL-2 and TNF-α in FYG-containing serum were also determined.

RESULTSFYG produced a potent antitumor effect (P<0.01) and induced marked apoptosis of the tumor tissue (P<0.05). Mice treated with FYG had higher percentages of CD3(+) and CD4(+) (P<0.05), and more NK cells (P<0.01) in the peripheral blood than those in the animals treated with normal saline. Mice receiving FYG had the highest serum levels of IL-2 and TNF-α (P<0.01). High-dose FYG-containing serum significantly decreased HepG2 cell viability, inhibited cell proliferation (P<0.05), and induced apoptosis (P<0.01). In addition, the levels of IL-2 and TNF-α of high-dose-containing serum were higher than the blank serum (P<0.01).

CONCLUSIONFYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

Animals ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; blood ; immunology ; pathology ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Hep G2 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Liver Neoplasms ; blood ; immunology ; pathology ; Lymphocyte Count ; Male ; Mice ; Mice, Inbred ICR ; Rats ; Serum ; Tumor Burden ; drug effects ; Tumor Necrosis Factor-alpha ; biosynthesis ; Xenograft Model Antitumor Assays

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.
Animals ; Antigens, CD/immunology/metabolism ; Bacterial Outer Membrane Proteins/*pharmacology ; Cells, Cultured ; Female ; Histocompatibility Antigens Class II/immunology/metabolism ; Host-Pathogen Interactions ; Interleukin-12/biosynthesis ; Interleukin-6/biosynthesis ; Legionella pneumophila/*immunology/metabolism ; Legionnaires' Disease/immunology/metabolism ; Lipoproteins/*pharmacology ; Macrophage Activation/drug effects/immunology ; Macrophages, Peritoneal/drug effects/immunology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Toll-Like Receptor 2/*metabolism ; Tumor Necrosis Factor-alpha/biosynthesis

The effect of extracellular beta-(1-->3), (1-->6)-glucan, produced by Paenibacillus polymyxa JB115, on nitric oxide (NO) production in RAW264.7 macrophages was investigated. beta-glucan induced the production of NO by RAW264.7 macrophages in a concentration- and time-dependent manner. Moreover, beta-glucan stimulation increased the mRNA expression of iNOS, COX-2 and IL-6 in RAW264.7 macrophages in a concentration-dependent manner.
Animals ; Bacillus/*metabolism ; Cell Line ; Cyclooxygenase 2/biosynthesis/genetics ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/*drug effects/enzymology/immunology ; Mice ; Nitric Oxide/*biosynthesis/immunology ; Nitric Oxide Synthase Type II/biosynthesis/genetics/metabolism ; RNA, Messenger/biosynthesis/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; beta-Glucans/metabolism/*pharmacology

OBJECTIVETo investigate the mechanisms that coagulation factor VIIa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro.

METHODSThe expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor VIIa or protease activated receptor 2 agonist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR.

RESULTSFactor VIIa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, upregulated TF mRNA expression and TF activity, but down-regulated caspase-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor VIIa were not only blocked by anti-TF but also by anti-PAR2 antibodies.

CONCLUSIONFactor VIIa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caspase-7 expression, thus promotes the tumor cell proliferation and migration. p38 MAPK may negatively regulate this process.

Caspase 7 ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; Interleukin-8 ; biosynthesis ; genetics ; Oligopeptides ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; agonists ; Thromboplastin ; biosynthesis ; genetics ; p38 Mitogen-Activated Protein Kinases ; metabolism

As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NSO cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3x10(6) cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (> 30 mmol/L). These results were important to provide technique and theory basis for developing optimized GS-NS0 cell culture and anti-CD25 monoclonal antibody production processes.
Antibodies, Monoclonal ; biosynthesis ; immunology ; Cell Line, Tumor ; Culture Media, Serum-Free ; pharmacology ; Culture Techniques ; methods ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; Multiple Myeloma ; metabolism ; pathology ; Recombinant Proteins ; biosynthesis ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo evaluate the effects of tramadol on the proinflammatory responses in a rat model of incisional pain by investigating its effects on nociceptive thresholds and serum interleukin-6 (IL-6) and IL-2 levels.

METHODSForty-two male Sprague-Dawley (SD) rats scheduled for plantar incision were randomly divided into 7 groups (n=6 in each group). Rats in Group 1 receiving general anesthesia with no incision were served as control; At 30 min before skin incision, Groups 2 to approximately 5 were given 5 ml normal saline or 1, 10, and 20 mg/kg tramadol, respectively, intraperitoneally (i.p.); Group 6 received 10 mg/kg tramadol after operation; Group 7 received 10 mg/kg tramadol before incision, followed by 200 microg/kg naloxone after operation. Mechanical allodynia was measured by electronic von Frey filament to evaluate the nociceptive thresholds 1 h before incision, and 1 h and 2 h after operation. Serum IL-6 and IL-2 levels were measured by enzyme-linked immunosorbent assay (ELISA) 2 h after operation.

RESULTSMechanical thresholds decreased significantly and serum IL-6 level increased significantly after operation in Group 2 compared with control (P<0.01), and these changes were reversed respectively by tramadol in a dose-dependent manner (P<0.05 and P<0.01, respectively). IL-2 level remained unchanged after operation in Group 2, but decreased in Group 3 (P<0.05), then gradually returned to the normal level in Groups 4 and 5. The intraperitoneally injected tramadol (10 and 20 mg/kg) produced a potent and dose-dependent antinocicptive effect on the lesioned paw. The antinocicptive effects of tramadol were partially antagonized by naloxone (200 microg/kg), suggesting an additional non-opioid mechanism.

CONCLUSIONThe results suggest that tramadol could be a good choice for the treatment of pain under the conditions that immunosuppression may be particularly contraindicated.

Analgesics, Opioid ; pharmacology ; Animals ; Dose-Response Relationship, Drug ; Interleukin-2 ; biosynthesis ; blood ; Interleukin-6 ; biosynthesis ; blood ; Male ; Pain Measurement ; methods ; Pain Threshold ; drug effects ; Pain, Postoperative ; blood ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tramadol ; pharmacology

OBJECTIVETo investigate the effect of trichostatin A (TSA) on proliferation and interleukin-2 (IL-2) expression of mouse T cells in mixed lymphocyte culture (MLC), and explore its effect on T cell-mediated immune response.

METHODSBALB/c and C57BL mouse MLC was treated with different concentrations of TSA for different durations, and the lymphocyte inhibition ratio was measured by MTT assay. With CTX as the control, one-way MLC system of BALB/c and C57BL mouse was treated with the same concentrations of TSA, and IL-2 expression in the T cells was observed by flow cytometry.

RESULTSTSA inhibited the proliferation of T cells in the MLC in a time- and dose-dependent fashion. It also reduced the IL-2 expression in one-way MLC dose-dependently, showing it significantly differed from the effect of CTX (P<0.01).

CONCLUSIONHistone deacetylase inhibitor TSA can inhibit the proliferation and reduce IL-2 expression of the T cells in MLC of mice, and therefore inhibit the T cell-mediated immune response.

Animals ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Female ; Flow Cytometry ; Histone Deacetylase Inhibitors ; Hydroxamic Acids ; pharmacology ; Interleukin-2 ; biosynthesis ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; cytology ; drug effects ; metabolism

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.
Anti-Inflammatory Agents/immunology/pharmacology ; Cell Line, Tumor ; Cyclic GMP/analogs & derivatives/immunology/metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors/metabolism ; Humans ; Interleukin-1beta/*metabolism ; Male ; Nitric Oxide/*biosynthesis/genetics/immunology ; Nitric Oxide Synthase Type II/*biosynthesis/genetics/immunology ; Phosphodiesterase Inhibitors/immunology/*pharmacology ; Piperazines/immunology/*pharmacology ; Purines/immunology/pharmacology ; Signal Transduction/drug effects/genetics/immunology ; Sulfones/immunology/*pharmacology ; Synovial Membrane/enzymology/immunology