We purified a novel mannose binding lectin form Musca domestica pupae by affinity chromatography on Con A-Sepharose 4B and DEAE weak anion-exchange chromatography. By SDS-PAGE, MBL-1 yielded a single band with the molecular weight of 24 kDa. It was a glycoprotein detected by periodic acid-schiffs staining reaction, with 97.36% protein and 2.1% oligosaccharide. Meanwhile, the results of beta-elimination reaction, infrared spectroscopy, atomic force microscopy and protein sequencing instrument show that MBL-1 was an ellipsoidal-shaped monomer with 60-100 nm in diameter. N-glycoside bond linked oligosaccharide chain and the N-terminal blocked peptide chain. Further study suggested that MBL-1 promote the proliferation of macrophage in a concentration-dependent manner. The scanning electron microscope analysis shows that MBL-1 promoted the activation of macrophages. These results show that MBL-1 purified from Musca domestica pupae possesses immune regulation effect, serving a reference basis to develop natural immune-modulator.
Animals ; Glycoproteins ; analysis ; Houseflies ; chemistry ; Immunomodulation ; immunology ; physiology ; Macrophages ; immunology ; Mannose-Binding Lectin ; chemistry ; physiology ; Oligosaccharides ; analysis ; Pupa ; chemistry

Glycyrrhizin acid and licorice flavonoids are the component of Glycyrrhiza uralensis Fisch root that has been used for various medicinal purposes in traditional oriental medicine for thousands of years. Macrophages as a principal component of immune system play an important role in the initiation, modulation and final activation of immune response against pathogens. In the present study, glycyrrhizin acid and licorice flavonoids was investigated the anti-inflammatory effect on lipopolysaccharide (LPS)-induced macrophage cell line of RAW264.7. Well-grown RAW264.7 cells were collected and randomly divided into the blank control group, the LPS(1 mg x L(-1)) group, the dexamethasone (5 mg x L(-1)) with LPS group, the glycyrrhizin acid (400, 80, 16 mg x L(-1)) with LPS group and the licorice flavonoids (200, 40, 8 mg x L(-1)) with LPS group. RAW264.7 cells were cultured in 24-well plates, pre-incubated for 4 h with different concentrations of dexamethasone, glycyrrhizin acid, or licorice flavonoids. Then cells were stimulated for 20 h with LPS. The supernatant of culture medium was collected from each well and determinated the concentrations of cytokines by means of BioPlex mouse cytokines assay. Compared with the control group, the LPS group could significantly induced relatively high levels of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor( GM-CSF), macrophage inflammatory protein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), regulated upon activation normal T cell expressed and secreted factor (RANTES), tumor necrosis factor alpha ( TNF-α), monocyte chemotactic protein 1 (MCP-1), chemokine (C-X-C motif) ligand 1 (KC), eotaxin, interleukin(IL)-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, and IL-17 secretion (P < 0.05). The glycyrrhizin acid significantly inhibited IL-1β, IL-3, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, Eotaxin and TNF-α secreted by LPS-stimulated RAW264.7 cells (P < 0.05). The expression levels of IL-6 and Eotaxin were observably decreased in the licorice flavonoids with LPS group (P < 0.05). The data presented here suggested that the glycyrrhizin acid and licorice flavonoids modulate various cytokines secreted by macrophages and were important anti-inflammatory constituent of Licorice.
Animals ; Cell Line ; Cytokines ; genetics ; immunology ; Flavonoids ; pharmacology ; Glycyrrhiza ; chemistry ; Glycyrrhizic Acid ; pharmacology ; Lipopolysaccharides ; immunology ; Macrophages ; drug effects ; immunology ; Mice

High mobility group box 1 (HMGB1) is an evolutionarily conserved non-histone chromatin-binding protein. During infection or injury, activated immune cells and damaged cells release HMGB1 into the extracellular space, where HMGB1 functions as a proinflammatory mediator and contributes importantly to the pathogenesis of inflammatory diseases. Recent studies reveal that inflammasomes, intracellular protein complexes, critically regulate HMGB1 release from activated immune cells in response to a variety of exogenous and endogenous danger signals. Double stranded RNA dependent kinase (PKR), an intracellular danger-sensing molecule, physically interacts with inflammasome components and is important for inflammasome activation and HMGB1 release. Together, these studies not only unravel novel mechanisms of HMGB1 release during inflammation, but also provide potential therapeutic targets to treat HMGB1-related inflammatory diseases.
HMGB1 Protein ; chemistry ; metabolism ; Humans ; Inflammasomes ; metabolism ; Macrophages ; immunology ; metabolism ; eIF-2 Kinase ; metabolism

The study aimed to explore the in vivo immunoregulatory function of Grifola frondosa polysaccharide( GFP) on animal disease models. Databases of PubMed,Embase,Web of Scinece,CNKI,CBM and Wan Fang Data were searched from the date of their establishment to February 2018. Two reviewers independently screened included studies and evaluated their quality by using SYRCLE's risk of bias tool. R software was used to analyze the data. Finally,20 animal experiment studies were included. According to Metaanalysis. For cellular immunity,GFP could effectively enhance the proliferation of effect or T cells,natural killer cells and macrophages in mice. The percentage of CD4+T cells( MD = 1. 89,95% CI [0. 94,2. 83],P < 0. 000 1),CD8+T cells( MD = 8. 46,95% CI[5. 93,11. 00],P<0. 000 1),NK cells( MD= 2. 67,95% CI [0. 23,5. 11],P= 0. 03),and macrophages( MD= 14. 09,95% CI[0. 84,27. 34],P= 0. 04) were all higher than those in control group. For humoral immunity,GFP could increase the secretion of TNF-α and INF-γ． The secretion of TNF-α( SMD = 15. 92,95% CI [9. 07,22. 76],P<0. 000 1) and INF-γ( SMD = 5. 34,95% CI[3. 42,7. 26],P<0. 000 1) were all higher than those in control group. In conclusion,GFP could regulate immunologic function by enhancing the proliferation activity of immune cells( CD4+T cells,CD8+T cells,NK cells and macrophages) and the secretion of immune factors( TNF-α and INF-γ) ． However,it is necessary to further standardize the selection of specific surface markers of immune cells and the administration of GFP,in order to reduce the heterogeneity among the studies. At the same time,more attention shall be paid to experimental design,implementation and full report,especially to the establishment and implementation of animal experimental registration system,so as to improve the transparency and quality of the whole process of animal experimental research,enhance the value of basic research ultimately,and provide a reliable theoretical basis for the transformation of basic research into clinical research.
Animals ; Cytokines/immunology* ; Disease Models, Animal ; Grifola/chemistry* ; Immune System ; Killer Cells, Natural/immunology* ; Macrophages/immunology* ; Mice ; Polysaccharides/pharmacology* ; T-Lymphocytes/immunology*

The role of alveolar macrophages (AMs) in the pathogenesis of asthma is still unknown. The aim of the present study was to investigate the effects of AM in the murine model of asthma. AMs were selectively depleted by liposomes containing clodronate just before allergen challenges, and changes in inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid were measured. AMs were then adoptively transferred to AM-depleted sensitized mice and changes were measured. Phenotypic changes in AMs were evaluated after in vitro allergen stimulation. AM-depletion after sensitization significantly increased the number of eosinophils and lymphocytes and the concentrations of IL-4, IL-5 and GM-CSF in BAL fluid. These changes were significantly ameliorated only by adoptive transfer of unsensitized AMs, not by sensitized AMs. In addition, in vitro allergen stimulation of AMs resulted in their gaining the ability to produce inflammatory cytokines, such as IL-1beta, IL-6 and TNF-alpha, and losing the ability to suppress GM-CSF concentrations in BAL fluid. These findings suggested that AMs worked probably through GM-CSF-dependent mechanisms, although further confirmatory experiments are needed. Our results indicate that the role of AMs in the context of airway inflammation should be re-examined.
Animals ; Asthma/*immunology ; Bronchoalveolar Lavage Fluid/chemistry/cytology/immunology ; Cytokines/biosynthesis/immunology ; Disease Models, Animal ; Female ; Immunization ; Immunomodulation/*immunology ; Inflammation/*immunology ; Leukocytes/immunology ; Macrophages, Alveolar/*immunology ; Mice ; Mice, Inbred C57BL ; Ovalbumin/immunology

Multi-components in herbal formulae exert holistic effects in synergistic or additive manners. However, appropriate strategies and supportive evidences are still lacking to uncover the synergistic or additive combinations. The present investigation aimed at seeking a screening strategy to identify the targeted combinations in GuGe FengTong Tablet (GGFTT), an herbal formula. Two compounds, belonging to different chemical classes, were combined with different concentration ratios and their anti-inflammation effects were investigated. The most significant anti-inflammatory combinations were evaluated by combination index (CI) method (additive effect, CI = 1; synergism, CI < 1; antagonism, CI > 1). The modulating effects of candidate combinations on pro-inflammatory cytokines and MAPKs signaling pathway were also detected. Two combinations, "biochanin A + 6-gingerol" (Bio-6G) and "genistein + 6-gingerol" (Gen-6G), showed synergistic effects (CI < 1), and Bio-6G was selected for further study. Compared with single compound, Bio-6G could synergistically inhibit the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and the activation of MAPKs signaling pathway in LPS-stimulated RAW264.7 cells. The combined results showed that Bio-6G was a synergistic anti-inflammatory combination in GGFTT. Our results could provide a useful strategy to screen the synergistic combinations in herbal formulae.
Animals ; Anti-Inflammatory Agents ; chemistry ; pharmacology ; Drug Compounding ; Drug Synergism ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Interleukin-1beta ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; immunology ; RAW 264.7 Cells ; Tablets ; chemistry ; pharmacology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo explore the pro-inflammatory toxicity of Pinellia pedatiecta, as well as the alum processing method on its pro-inflammatory effect.

METHODRaphide and agglutinin (PPA) proteins were isolated from fresh P. pedatiecta. The overall animal and cellular level models were applied to investigate the pro-inflammatory effect of raphide and PPA in P. pedatiecta, as well as the impact of the alum processing method on the pro-inflammatory effect, with inflammatory mediators as the index.

RESULTIntraperitoneal injection with P. pedatiecta raphide suspension could significantly increase the content of inflammatory mediators PGE2 and NO. After the alum processing method was adopted, fresh P. pedatiecta and raphide-induced PGE2 and NO release significantly reduced. The stimulation of mice macrophages with P. pedatiecta agglutinin protein could cause the content of dose-dependent inflammatory mediators TNF-alpha and IL-6. After the alum processing method was adopted, PGE2 content in P. pedatiecta agglutinin protein-induced mice peritoneal exudate notably decreased.

CONCLUSIONThe irritation and toxicity of P. pedatiecta were inflammatory responses in organisms. Its raphide and agglutinin proteins were toxic components, both could cause significant the release of inflammatory medium. The alum processing method could help significantly reduce the pro-inflammatory toxicity of P. pedatiecta.

Alum Compounds ; chemistry ; Animals ; Drug Compounding ; methods ; Drugs, Chinese Herbal ; chemistry ; toxicity ; Female ; Inflammation Mediators ; chemistry ; toxicity ; Interleukin-6 ; immunology ; Macrophages ; drug effects ; immunology ; Male ; Mice, Inbred ICR ; Pinellia ; adverse effects ; chemistry ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo examine the anti-inflammatory mechanism of total flavonoids of Glycyrrhizae Radix et Rhizoma (TFGR) and its ingredient on IFN-gamma and LPS-induced macrophage RAW264.7.

METHODSolvent extraction and macroporous resin enrichment were adopted for preparing ethanol extracts of Glycyrrhizae Radix et Rhizoma, components and total flavonoids. Ultraviolet spectroscopy was used to determine the content of total flavonoids. An IFN-gamma and LPS-induced cell inflammatory model was established. Griess reaction was used for detecting the effect of extracts at all levels and flavonoid monomers on nitrite content in cell culture supernatant. FRAP was used for measuring anti-oxidation capacity. RT-PCR was used for determining the effect of TFGR and isoliquiritigenins on intracellular inducible nitric oxide synthase iNOS, COX-2, IL-6 and PPAR-gamma. Western blot was used for detecting the effect of TFGR and isoliquiritigenins on iNOS, COX-2 and MAPK signal transduction pathways.

RESULTCompared with other extracts, ethyl acetate fractions from Glycyrrhizae Radix et Rhizoma showed the highest inhibition ratio on nitrite content at the same concentration. After being enriched with macroporous resin, TFGR (60. 08% of liquiritin) of ethyl acetate extracts from Glycyrrhizae Radix et Rhizoma showed dose-dependence, and inhibited the nitrite content in cell culture supernatant, which was superior to ethyl acetate extracts, and had the protective effect on post-stimulated cell activity, with a stronger total anti-oxidation than other extracts. TFGR inhibited iNOS, IL-6 mRNA, protein expressions of iNOS, COX-2 and IL-6. Isoliquiritigenin, a flavonoid monomer, could inhibited iNOS, COX-2 gene and protein expression and gene expressions of IL-1beta and IL-6, and upside-regulated gene expression of PPAR-gamma.

CONCLUSIONActivity-oriented extraction suggests that ethyl acetate fractions from Glycyrrhizae Radix et Rhizoma is one of components with anti-inflammatory activity. TFGR obtained by enriching the active component showed dose-dependence, and inhibited the nitrite content in cell culture supernatant. The anti-inflammatory effect is partially achieved by regulating ERK signal pathway and inhibiting iNOS and COX-2 gene and protein expressions through extracellular signals of mitogen activated protein kinases (MAPKs). Specifically, isoliquiritigenin may be a component with TFGR anti-inflammatory activity.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Glycyrrhiza ; chemistry ; Interleukin-1beta ; genetics ; immunology ; Interleukin-6 ; genetics ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Rhizome ; chemistry

Radix Adenophorae, a traditional Chinese medicine, has been reported to have a variety of biological functions. In the present study, a polysaccharide component, Radix Adenophorae Polysaccharide (RAPS), was purified from Radix Adenophorae by decoloring with ADS-7 macroporous adsorption resin, DEAE-52 cellulose ion-exchange chromatography, and Sephacryl S-300HR gel chromatography, with the purity of 98.3% and a molecular weight of 1.8 × 10(4) Da. The cell viability assay and microscopic examination revealed that RAPS promoted the proliferation and activation of macrophages. At 400 μg·mL(-1), RAPS stimulated RAW264.7 cell proliferation by 1.91-fold compared with the control. Meanwhile, RAPS significantly increased the secretion of pro-inflammatory cytokines (TNF-α and IL-6) in a dose-dependent manner in the supernatant of RAW264.7 cell culture as determined by ELISA. At 400 μg·mL(-1), the production of TNF-iα was 20.8-fold higher than that of the control. Simultaneously, the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) were increased in RAW264.7 cells incubated with RAPS, as measured by Griess assay and Western blot analysis. The NO production of cells treated with RAPS (400 μg·mL(-1)) reached 15.8 μmol·L(-1), which was 30.4-fold higher than that of the control (0.53 μmol·L(-1)). These data suggested that RAPS may enhance the immune function and protect against exogenous pathogens by activating macrophages.
Animals ; Campanulaceae ; chemistry ; Cytokines ; genetics ; immunology ; Immunologic Factors ; pharmacology ; Interleukin-6 ; genetics ; immunology ; Macrophage Activation ; drug effects ; Macrophages ; drug effects ; immunology ; Mice ; Nitric Oxide ; immunology ; Plant Extracts ; pharmacology ; Polysaccharides ; pharmacology ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVETo observe the effects of bagasse polysaccharide on the immune functions of immunosuppressed mice.

METHODSImmunosuppressed mouse models were established by intraperitoneal injections with cyclophosphamide followed by daily intragastric administration of bagasse polysaccharide. After the treatments, the mice were examined for immune organ weight index, phagocytotic function of the macrophages, delayed type hypersensitivity, serum IgM level following exposure to chicken red blood cells, formation of hemolytic plaques, T cell percentage and lymphocyte transformation.

RESULTSTreatment of the immunosuppressed mice with bagasse polysaccharide at the daily dose of 200 and 400 mg/kg significantly increased the weight of the immune organs, phagocytotic function of the macrophages, delayed type hypersensitivity, serum IgM level against chicken red blood cells, formation of hemolytic plaques, T cell percentage and lymphocyte transformation.

CONCLUSIONBagasse polysaccharide can enhance the immune functions of immunosuppressed mice.

Animals ; Cellulose ; chemistry ; Cyclophosphamide ; Female ; Immunocompromised Host ; immunology ; Lymphocyte Activation ; drug effects ; Macrophages ; immunology ; Male ; Mice ; Phagocytosis ; drug effects ; Polysaccharides ; pharmacology ; Random Allocation