Cyclovirobuxinum D suppresses lipopolysaccharide-induced inflammatory responses in murine macrophages in vitro by blocking JAK-STAT signaling pathway.

AIM: Cyclovirobuxinum D (CVB-D), an alkaloid isolated from the Chinese medicinal plant Buxus microphylla, has been found to be effective to treat cardiac insufficiency, arrhythmias and coronary heart disease. In the present study, we investigated the effects of CVB-D on the inflammatory responses in lipopolysaccharide (LPS)-stimulated murine macrophages in vitro and the underlying mechanisms. METHODS: Murine macrophage cell line RAW264.7 cells were incubated in the presence of LPS (0.1 µg/mL) for 24 h. The cell viability was measured using MTT assay. The release of NO and cytokines were detected using the Griess test and ELISA, respectively. The mRNA and protein levels were determined using RT-PCR and Western blot, respectively. Reporter gene assays were used to analyze the transcriptional activity of NF-κB. RESULTS: Treatment of RAW264.7 cells with CVB-D (25-300 µmol/L) did not affect the cell viability. Pretreatment with CVB-D (50, 100 and 200 µmol/L) concentration-dependently decreased NO release and iNOS expression in LPS-treated RAW264.7 cells (its IC50 value in inhibition of NO production was 144 µmol/L). CVB-D also concentration-dependently inhibited the secretion and mRNA expression of IL-1ß and IL-6 in LPS-treated RAW264.7 cells. Furthermore, CVB-D remarkably inhibited the phosphorylation of STAT1 and STAT3, as well as JAK2 in LPS-treated RAW264.7 cells, but did not affect the activation of NF-κB and MAPKs pathways. Pretreatment with the JAK2 specific inhibitor AG490 (30 µmol/L) produced similar effects on NO release and iNOS expression in LPS-treated RAW264.7 cells. CONCLUSION: CVB-D exerts anti-inflammatory effects in LPS-stimulated murine macrophages in vitro at least in part by blocking the JAK-STAT signaling pathway. The anti-inflammatory actions of CVB-D may contribute to its cardioprotection.

[Study on the anti-endotoxin effect of saponin from Tupistra chinensis].

OBJECTIVE: To investigate the effect of saponin from Tupistra chinensis Baker (STCB) on lethal toxicity of endotoxin in mice and explore the underlying mechanism. METHODS: Mouse models of endotoxin-induced death and endotoximia were established by intraperitoneal administration of KM mice with lipopolysaccharides (LPS) from Pseudomonas aeruginosa in doses of 60 mg/kg and 10 mg/kg respectively. Mouse survival rate and survival time were recorded and the serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in endotoximia mice were measured by enzyme-linked immunosorbent assay (ELISA). Mouse peritoneal exudate cells induced by LPS were used as an in vitro inflammatory model,which was then intervened with STCB and the levels of IL-1beta and TNF-alpha in the culture supernatants were measured by ELISA. RESULTS: The survival rates of mice prophylactically treated with STCB (200 and 400 mg/kg, in 5 consecutive days) were slightly higher compared with that in model group,but no statistical difference was observed (P>0.05). The survival time was much longer in the treated group (P<0.05). The serum levels of IL-1beta and TNF-alpha in STCB-treated mice (200 and 400 mg/kg, in 5 consecutive days) were significantly lower compared with those in model group (P<0.05). STCB (20 and 40 microg/mL) remarkably inhibited LPS-induced IL-1beta and TNF-alpha production by peritoneal exudate cells in vitro (P<0.05). CONCLUSION: Saponin from Tupistra chinensis showed beneficial effect on the prevention of mice from lipopolysaccharides-induced death, in which down regulation of IL-1beta and TNF-alpha expression might be involved.

[Process optimization for extraction of immune active polysaccharides from Fomes fomentarius by introduction of ultrasonication].

OBJECTIVE: To study the optimal extraction process of immune active polysaccharides from Fomes fomentarius by introduction of ultrasonication. METHODS: An orthogonal experimental design of L9 (3(4)) was used to investigate the effects of ultrasonication time, extraction temperature and extraction time on the extraction ratio, sugar content and immune stimulating activity (mouse splenocyte metabolic activity measured with MTT colorimetry) of the polysaccharides and the optimal extraction process was evaluated. RESULTS: Ultrasonication treatment had the most remarkable effect on the immune stimulating activity of the polysaccharides. The optimal extraction process for extraction of immune active polysaccharides was as follows: ultrasonication for 30min, extraction temperature at 80 degrees C and water extraction time for 2h. CONCLUSION: Ultrasonication can be used as a useful technique for extraction of immune active polysaccharides from Fomes fomentarius.

[Effect of Flammulina velutipes polysaccharides on production of cytokines by murine immunocytes and serum levels of cytokines in tumor-bearing mice].

OBJECTIVE: To study the effect of Flammulina velutipes polysaccharides (FVP) on the production of tumor necrosis factor alpha (TNF-alpha), interferon-gamma (INF-gamma) and interleukin 2 (IL-2) by murine immunocytes. METHODS: The cell's metabolic activity was determined with methylthiazolyl tetrazolium (MTT) colorimetry assay and the amounts of TNF-alpha, INF-gamma and IL-2 were measured by ELISA. RESULTS: FVP (200, 100, 50 microg/mL) could promote the metabolic activity of murine splenocytes and peritoneal exudate cells (PEC) and increase the amounts of TNF-alpha, INF-gamma and IL-2 in the supernatants of splenocyte cultures, and the amount of TNF-alpha in PEC cultures, with the most marked increase on TNF-alpha level. FVP (100, 50, 25 mg/kg) could raise the serum levels of TNF-alpha and INF-gamma in S180 tumor-bearing mice. CONCLUSION: FVP may regulate murine immune function through promoting the production of TNF-alpha, INF-gamma and IL-2.

[Study of anticoagulant activity of ethanol extracts from leech in vitro].

OBJECTIVE: To study the anticoagulant activity of different portions of leech ethanol extracts (LEEs). METHODS: Anticoagulant activity was determined by measuring prothrombin time(PT), thrombin time (TT), activated partial throboplstin time (APTT) and fibrinogen coagulation time (FCT). RESULTS: PT, TT, APTT and FCT were remarkably prolonged by ethyl acetate portion of LEEs. Portions of petroleum ethrer, n-butanol and water extracted from LEEs was much weaker on anticoagulant activity. CONCLUSION: The anticoagulant effect of ethyl acetate extract portion of LEEs is the strongest among the four portions, and this results may be from its inhibitory effect on thrombin-catalyzed fibrinogen hydrolysis.

[Purification, identification of acoagulatin, an anticoagulation factor from the venom of Chinese Agkistrodon, and observation of its anticoagulation effect].

OBJECTIVE: To isolate and identify an anticoagulation factor, acoagulatin, from the venom of Chinese Agkistrodon and to observe its anticoagulation effect. METHOD: The venom of Chinese Agkistrodon was isolated and purified using ion-exchange chromatography on DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow. High-performance liquid chromatography (HPLC) was performed for determination of the purity of acoagulatin, and SDS-PAGE electrophoresis (with 5% concentrated gel of pH 6.8, 12% separation gel of pH 8.8, Tris-aminoacetic acid buffer of pH 8.3 as the electrode buffer) for determining the relative molecular mass. For observation of the anticoagulation effect, 20 microl acoagulatin solution at the concentration of 0.30, 0.20 and 0.15 microg/microl, respectively, was mixed with 100 microl rabbit anticoagulated plasma and the thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined. RESULTS: Acoagulatin was found to consist of two subunits with relative molecular mass of 14 400 and 17 000 respectively, resulting in the total relative molecular mass of 31 400 as determined by SDS-PAGE. HPLC demonstrated good homogeneity of this protein, which significantly prolonged the PT and APTT without affecting TT. CONCLUSION: DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow ion exchange column chromatographies are effective to isolate acoagulatin of high purity, which possesses anticoagulation effect.

[Immunomodulatory effects of Fomes fomentarius polysaccharides: an experimental study in mice].

OBJECTIVE: To investigate the immunomodulatory effects of Fomes fomentarius polysaccharides (FFP) in mice. METHODS: MTT assay was employed to evaluate the in vitro metabolic activity of the mouse splenocytes treated with FFP at different concentrations, and the secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and interleukin 2 (IL-2) from the cells were measured by enzyme-linked immunosorbent assay. The changes in the phagocytotic activity of mouse macrophage in response to FFP treatment were evaluated by phagocytosis percentage of chicken red blood cells (CRBCs). The effect of FFP on the humoral immunity was assessed in mice immunized with sheep red blood cells (SRBCs) by measuring the serum levels of specific antibody (hemolysin) against SRBCs. RESULTS: FFP at the concentrations of 25, 50, and 100 microg/ml all significantly enhanced the metabolic activity of mouse splenocytes in vitro and increased the production of TNF-alpha, IFN-gamma and IL-2. FFP treatment also markedly enhanced the metabolic activity of mouse peritoneal exudate cells and TNF-alpha production by the cells. At the doses of 25, 50, and 100 mg/kg, FFP significantly increased serum hemolysin level in mice immunized with SRBCs, and FFP at 50 and 100 mg/kg obviously increased the capacity of mouse peritoneal macrophages in vivo for CRBC phagocytosis. CONCLUSION: FFP can promote the secretion of TNF-alpha, IFN-gamma and IL-2 by mouse immunocytes and enhance mouse humoral immune response and the phagocytotic activity of the macrophages.

[Effects of lipopolysaccharides of Bacterium prodigiosum on tumor growth and immunosuppression in mice].

OBJECTIVE: To observe the effects of lipopolysaccharides of Bacterium prodigiosum (BP-LPS) in inhibiting tumor growth and improving immunosuppression in mice. METHODS: In mice bearing S180 tumor and a mouse model of immunosuppression induced by cyclophosphamide (CTX), the tumor growth, indexes of the immune organs and peripheral white blood cell count were measured after intraperitoneal injection of BP-LPS. RESULTS: Injections of BP-LPS (40 U/kg) for 8 consecutive days resulted in a significant inhibition of the tumor growth in mice bearing S180 tumor (P<0.01), with a dose-dependent increase of the spleen indexes but no obvious changes in the thymus indexes. Intraperitoneal injections of BP-LPS for 7 days inhibited the reduction of peripheral white blood cells and spleen indexes in immunosuppressive mice, but did not produce any significant changes in normal mice. CONCLUSION: BP-LPS can inhibit the tumor growth in tumor-bearing mice and enhance the immune functions of immunosuppressive mice.

[Establishment of mouse model of humoral immune response using rabbit red blood cells as the antigen].

OBJECTIVE: To establish a mouse model of humoral immune response by immunization with rabbit red blood cells (RRBCs). METHODS: The mice were immunized with RRBCs and the serum hemolysin level was measured by micro-hemolysis spectrophotometry. RESULTS: The peak time needed for hemolysin production against RRBCs was 6 days after the immunization, and 20% RRBCs in a total volume of 0.2 ml was optimal for intraperitoneal injection. Hydrocortisone (25 mg/kg) and cyclophosphamide (20 mg/kg) inhibited hemolysin production. Mannatide (4 mg/kg) produced no significant effect on serum hemolysin level in normal mice, but significantly potentiated hemolysin production in immunosuppressed mice induced by cyclophosphamide (20 mg/kg). CONCLUSION: Intraperitoneal RRBC injection is feasible for establishing mouse models of humoral immune response.

[Changes of serum interferon-gamma levels in mice bearing S-180 tumor and the interventional effect of immunomodulators].

OBJECTIVE: To investigate the changes of serum inteferon-gamma (IFN-gamma) in mice bearing S-180 tumor and explore the role of the endogenous IFN-gamma in confining the transplanted tumor by intervention with immunomodulators. METHODS: Mouse models bearing S-180 solid tumor were established and subjected to intragastric administration of Ganoderma lucidum polysaccharides (GLP) or cyclosporine A (CsA) at different daily doses for 9 consecutive days. Serum IFN-gamma levels were measured in untreated tumor-bearing mice and in those after completion of GLP or CsA treatments by enzyme-linked immunosorbent assay (ELISA), and the changes of the tumor weight in the treated mice were evaluated. RESULTS: It was found for the first time that serum IFN-gamma levels in the tumor-bearing mice increased progressively within the initial 20 days after tumor implantation. The serum IFN-gamma levels in the 3 GLP-treated groups (at daily doses of 400, 200, and 100 mg/kg) all increased, which was the most obvious in 400 mg/kg GLP-treated group, and the tumor weight decreased significantly in response to GLP treatment, but the most conspicuous effect occurred with the daily dose of 200 mg/kg, and no significant statistical correlation was found between the two parameters. CsA treatment (at 20, 10, and 5 mg/kg, respectively) resulted in reduced serum IFN-gamma levels but produced virtually no effect on the tumor weight, and no obvious correlation was found between serum IFN-gamma level and the tumor weight. CONCLUSION: Increased serum IFN-gamma levels following GLP treatment are not significantly correlated to tumor growth inhibition in mice, and CsA reduces serum IFN-gamma levels without affecting the tumor weight, suggesting that endogenous IFN-gamma is not a major immunomodulating factor in growth inhibition of transplanted S-180 tumor.