OBJECTIVETo establish flow cytometry (FCM) methods and evaluate their application value for measuring the index for enhancing immune function of health food.

METHODSIn mice experiment model, the dosage groups were respectively oral fed with three test substances according to 5, 10, 30 times of the recommended dose for human body; both the negative and positive control groups were fed with equivalence purified water once a day. The positive control was fed with 25 mg/kg body weight levamisole for 3 days before finishing the administration, and the immune two percent of sheep erythrocytes were administrated at the last day. In rats experiment model, the test substance was given by mixing feed according to 25 and 50 times of the recommended dose for human body. At the end of the experiment, indices below were simultaneously detected. (1) The classical indices included: spleen lymphocyte transformation test by using ConA (MTT assay); spleen NK cell activity test (LDH assay); delayed-type hypersensitivity test by using sheep erythrocyte (foot palm thickening) method and phagocytosis activity tested by mice peritoneal macrophages. (2) FCM indices included: T and B lymphocytes quantitating in mice peripheral blood, activated antigen expression level in the surface of T lymphocytes and NK cells and phagocytosis activity for fluospheres in mice peritoneal macrophages.

RESULTS(1) Compared with the negative control group, there were no significant differences in T and B lymphocytes proportion and the number of lymphocytes in mice peripheral blood after given 0.83, 1.67, 5.01 g/kg protein powder; (2) mice peripheral blood T lymphocyte sub-cluster CD(69)(+)/CD(3)(+) of 3.75, 7.50, 15.0 ml/kg bw Cen-Rong Cream groups were all significantly increased (P < 0.05), which were shown a good coherence with the classic test index; (3) mice peripheral blood NK cell sub-cluster CD(69)(+)/NKG2D(+) of 0.83, 1.67 g/kg protein powder groups were both significantly increased (P < 0.05), which was kept in good coherence with those of NK cell activity test (LDH assay); rats peripheral blood NK cell sub-cluster CD(161a+)/CD(25)(+) of 1.50 g/kg ganoderma lucidum and cordycepicmycelia group was significantly increased (P < 0.05); (4) the phagocytosis activity in mice peritoneal macrophages: there were no significant difference found between the controls and the dosage groups in the classic test. However, in the FCM test, the percentage of phagocytic cells of 0.15, 0.30, 0.90 g/kg ganoderma lucidum and cordycepicmycelia groups and the phagocytic index of 0.30, 0.90 g/kg were enhanced.

CONCLUSIONIt suggests that was shown in detecting and assessing enhancing immune function of health food the results tested by FCM were fairly consistent with those by using traditional methods, most of them would have higher sensitivity. It should be valuable to applying FCM in the measurement and assessment of enhancing immune function of health food and worth while to further study as to enlarging its application.

Animals ; Antigens, CD ; analysis ; Antigens, Differentiation, T-Lymphocyte ; analysis ; B-Lymphocytes ; cytology ; immunology ; CD3 Complex ; analysis ; Cell Survival ; Erythrocytes ; cytology ; immunology ; Female ; Flow Cytometry ; methods ; Food, Organic ; Humans ; Lectins, C-Type ; Macrophages, Peritoneal ; cytology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Rats ; Rats, Sprague-Dawley ; Sheep ; Spleen ; cytology ; T-Lymphocytes ; cytology ; immunology

OBJECTIVETo study the effect of Angelica sinensis, Salvia miltiorrhiza and Ligustrazine on function of peritoneal macrophages during peritoneal dialysis.

METHODSPeritoneal macrophages of mice were cultured in culture medium (control), peritoneal dialysate (PD), drugs contained PD containing Angelica, Salvia and Ligustrazine combined (PD-ASL) or separated (PD-A, PD-S, PD-L) with concentration of 2 micrograms/ml, 10 micrograms/ml and 100 micrograms/ml, separately for 24 hrs. The nitric oxide (NO) content, methyl thiazolyl tetrazolium (MTT) reducing capacity (MTT-RC) and phagocytosis capacity of macrophages were determined and compared.

RESULTSNO content and MTT-RC of macrophages cultured in PD group were significantly lower than those of the control (P < 0.01), as compared with those in drug contained PD groups, the NO content in the PD-L group and the MTT-RC in the PD-ASL group were higher significantly (P < 0.01). The phagocytosis capacity and NO content in the PD-ASL group were raised along with the increased concentration of drug in PD.

CONCLUSIONAdministering Chinese herbal medicine during peritoneal dialysis has important significance in improving the defense function of peritoneal macrophages, reducing the incidence of peritonitis and enhancing the therapeutic effect of peritoneal dialysis.

Angelica sinensis ; Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Macrophages, Peritoneal ; cytology ; immunology ; Male ; Mice ; Nitric Oxide ; metabolism ; Peritoneal Dialysis ; adverse effects ; Phagocytosis ; drug effects ; Phytotherapy ; Pyrazines ; pharmacology ; Salvia miltiorrhiza

OBJECTIVETo explore the influence of bone marrow (BM) mesenchymal stem cells (MSCs) on macrophage activation after lipopolysaccharide (LPS) stimulation.

METHODSMouse BM MSCs were isolated and purified by adherence screening, and mouse peritoneal macrophages (MPM) were collected by sodium thioglycollate peritoneal injection, and the co-culture system was established by planting macrophages on the MSCs monolayer. The grouping of experiments: group A: MPM; group B: MPM + LPS; group C: MPM + LPS + MSC; group D: MPM + LPS + MSC supernatant. Cell culture supernatants were collected to detect the changes of TNF-alpha/TGF-beta and nitrogen monoxide (NO) after stimulating macrophages with LPS for 18 hours. At the same time Escherichia coli standard strain (ATCC25922) was added into the culture system and incubated for another 24 hours, macrophages were stained and phagocytosis were examined.

RESULTSThe concentrations of TNF-alpha and NO in culture supernatants were increased significantly to (147.4 +/- 37.1) pg/ml and (59.9 +/- 8.7) micromol/L respectively after macrophage activation, however, at the present of MSC, the concentration of TNF-alpha was dramatically decreased [(97.6 +/- 30.3) pg/ml, P = 0.032], and the concentration of NO was decreased to (50.9 +/- 29.5) micromol/L (P > 0.05). The concentrations of TNF-alpha and NO were further decreased after addition of MSC supernatants [(58.3 +/- 31.5) pg/ml and (-3.4 +/- 2.3) micromol/L respectively, P < 0.01]. There was no change in the phagocytic rate and phagoindex of macrophages after activation.

CONCLUSIONSMSCs can inhibit the activation of mouse peritoneal macrophages after stimulating with LPS but has no influence on the phagocytosis.

Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Lipopolysaccharides ; pharmacology ; Macrophage Activation ; drug effects ; Macrophages, Peritoneal ; immunology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study effects of polysaccharide of Radix Ranunculi Ternati (PRT) on immunological function and anti-oxidation activity of mouse.

METHODCell proliferations of splenocyte, thymocyte and peritoneal macrophage were measured by MTT colorimetry. The phagocytic function of peritoneal macrophage was measured by neutral red colorimetric method. The disoxidation power of PRT was measured by Prussian blue method. The clearing effect of PRT on hydroxyl radical was measured by salicylic acid capture method. The clearing effect of PRT on superoxide anion free radical was measured by pyrogallol auto oxidation method.

RESULTPRT among 25-400 mg x L(-1) could enhance thymocytes and spleen lymphocyte proliferation and macrophage phagocytosis. PRT(200 mg x L(-1)) has the strongest macrophage proliferation. PRT in different concentration has shown some disoxidation effects. PRT in 8 g x L(-1) has nearly the same ability of clearing x OH by Vit C with the same concentration. The clearance rate of PRT on O2*- is 95.39%.

CONCLUSIONPRT can enhance the cell proliferation capability of thymocytes, spleen lymphocytes and peritoneal macrophages. PRT can enhance macrophage phagocytosis in a dose-response relationship. PRT has saome disoxidation power and strong ability of clearing x OH and O2*-.

Animals ; Antioxidants ; analysis ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Macrophages, Peritoneal ; cytology ; drug effects ; immunology ; Male ; Mice ; Oxidation-Reduction ; drug effects ; Phagocytosis ; drug effects ; Polysaccharides ; immunology ; pharmacology ; Ranunculus ; chemistry ; Spleen ; cytology ; drug effects ; immunology ; Thymus Gland ; cytology ; drug effects ; immunology

OBJECTIVETo study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene.

METHODSMurine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR. After mice were orally immunized with SL7207(pCMV-beta), the expression of beta-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test.

RESULTSThe results indicated that the plasmid pCMV-beta could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated S.typhimurium SL7207 harboring plasmid pCMV-beta mice, the expression of beta-gal could be detected in the spleen, mesenteric lymph nodes and Peyers patches of the mice. Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice. Compared with the naked DNA vaccination, SL7207 (pCMV-beta) oral immunization were more efficient in inducing cellular immune responses.

CONCLUSIONSAttenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response.

Animals ; Bacterial Vaccines ; immunology ; Cell Proliferation ; Cytotoxicity, Immunologic ; Female ; Genes, Reporter ; Genetic Vectors ; Macrophages, Peritoneal ; metabolism ; microbiology ; Mice ; Mice, Inbred BALB C ; Mutation ; Salmonella typhimurium ; genetics ; T-Lymphocytes, Cytotoxic ; cytology ; Transfection ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; beta-Galactosidase ; genetics ; immunology ; metabolism

The studies were conducted on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. Prior to restraint stress the mice were treated with sodium diethyldithiocarbamate (DTC) i.p. at a dose of 20 mg/kg five times at 48 h intervals. DTC was used per se or with zinc ions interaction, by adding zinc sulfate to drinking water at a dose of 72 microgram/mouse daily. The results obtained in the study show that restraint stress causes involution of lymphatic organs, decreased the percentage of immature (CD4+CD8+) and, mature (CD4+) thymocytes and CD4+, CD8+and CD19 + splenocytes and proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA). The restraint stress decreased also interleukin-1 (IL-1) production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from E. coli. Pretreatment with DTC counteracted restraint stress-induced immunosuppression, which is expressed as partial normalisation of the total number of thymocytes, splenocytes and IL-1 production, accelerated regeneration of thymus and spleen, shorter suppressive action of restraint stress on the percentage of CD4+CD8+thymocytes and in total normalisation of the CD4+thymocytes and splenocytes. DTC administered prior to restraint stress augmented the proliferative response of thymocytes to two mitogens. The immunocorrecting action of DTC is enhanced by zinc supplementation, expressed in the increased percentage of CD4+thymocytes and splenocytes, CD19 + splenocytes, proliferative activity of thymocytes stimulated with PHA and IL-1 production. The obtained results show that DTC administration can be supplemented with zinc in order to restore the immune system impaired by stress.
Adjuvants, Immunologic/*pharmacology ; Animals ; Ditiocarb/*pharmacology ; Female ; Immunity, Cellular/*drug effects ; Interleukin-1/biosynthesis ; Macrophages, Peritoneal/immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mitogens/biosynthesis ; Organ Size/drug effects ; Restraint, Physical ; Spleen/cytology/drug effects ; Stress/etiology/*immunology ; T-Lymphocyte Subsets/drug effects ; Thymus Gland/cytology/drug effects ; Zinc Sulfate/*pharmacology

OBJECTIVETo study the effects of heat-killed Penicillium marneffei (PM) on the expressions of toll-like receptor-4 (TLR-4), toll-like receptor-2 (TLR-2) and dendritic cell associated C-type lectin-1 (Dectin-1)and the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). in mouse peritoneal macrophages.

METHODSMouse peritoneal macrophages were cultured in the presence of heat-killed yeast-phase PM for 24 h, and the average fluorescence intensity of TLR-2, TLR-4, and Dectin-1 in the macrophages was detected using flow cytometry. Fluorescent staining of the macrophages was performed to observe the fluorescence of TLR-2, TLR-4, and Dectin-1 with confocal microscopy. TNF-alpha mRNA in the cell culture supernatant was measured with real-time PCR, and TNF-alpha protein detected using enzyme-linked immunosorbent assay (ELISA).

RESULTSThe average fluorescence intensity of TLR-2, TLR-4 and Dectin-1 in the macrophages was increased in response to a 24-h PM stimulation, and the stimulated macrophages produced large amounts of TNF-alpha.

CONCLUSIONPM up-regulates the expression of TLR-2, TLR-4 and Dectin-1 in mouse peritoneal macrophages, and their expressions are directly associated with macrophage activation.

Animals ; Cells, Cultured ; Lectins, C-Type ; Macrophages, Peritoneal ; cytology ; immunology ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins ; biosynthesis ; Penicillium ; immunology ; Toll-Like Receptor 2 ; biosynthesis ; Toll-Like Receptor 4 ; biosynthesis ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA).

METHODWister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated.

RESULTWuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity.

CONCLUSIONThe anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.

Animals ; Arthritis, Experimental ; drug therapy ; immunology ; metabolism ; pathology ; Capsules ; Dehydroascorbic Acid ; analogs & derivatives ; blood ; Dinoprostone ; metabolism ; Disease Models, Animal ; Edema ; drug therapy ; Female ; Interleukin-1beta ; metabolism ; Lymphocytes ; immunology ; metabolism ; Macrophages, Peritoneal ; metabolism ; Male ; Medicine, Mongolian Traditional ; Mice ; Nitric Oxide ; metabolism ; Rats ; Spleen ; cytology ; metabolism ; Superoxide Dismutase ; blood ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo observe the effect of andrographolide on the activation of mitogen-activated protein kinases (MAPKs) and expression of nuclear factor-κB (NF-κB) in macrophage foam cells.

METHODSThe mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein (ox-LDL), ox-LDL+andrographolide, or neither (control). The phosphorylation of MAPK molecules (p38MAPK, JNK, ERK1/2) and the expressions of NK-κB p65 were examined by Western blot.

RESULTSAs compared with cells in the control group, the expressions of phospho-p38 and NF-κB p65 were increased in the cells cultured with either ox-LDL or ox-LDL+andrographolide (P<0.01), but attenuated significantly in the presence of ox-LDL+ andrographolide when compared with ox-LDL (P<0.05). The phospho-JNK increased in the presence of either ox-LDL or ox-LDL+andrographolide when compared with control cells (P<0.01), but no significant difference existed between ox-LDL and ox-LDL+andrographolide (P>0.05). The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells (P<0.01), but no significant differences existed between the cells cultured in the presence of ox-LDL+andrographolide and the control medium (P>0.05).

CONCLUSIONSAndrographolide could inhibit the activation of ERK1/2, p38MAPK and NK-κB induced by ox-LDL in macrophage foam cells, which might be one of its mechanisms in preventing atherosclerosis.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Atherosclerosis ; immunology ; metabolism ; prevention & control ; Cells, Cultured ; Diterpenes ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Foam Cells ; cytology ; drug effects ; enzymology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lipoproteins, LDL ; metabolism ; MAP Kinase Signaling System ; drug effects ; immunology ; Macrophages, Peritoneal ; cytology ; drug effects ; enzymology ; Mice ; Mice, Inbred Strains ; NF-kappa B ; metabolism ; Vasculitis ; drug therapy ; immunology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo observe effect of Xue Hanjing oral fluid on mice immunological function.

METHODThe weight of thymus gland and spleen and the function of abdominal cavity macrophage were measured. Production of the hemolysin antibody, the immunoglobulin of blood serum and complement and the proliferation of T lymphocytes were observed respectively by means of microblood, immune-turbidimetry and MTT staining.

RESULTXue Hanjing oral fluid could enhance index of the thymus gland and spleen and the phago-percent of abdominal cavity macrophage, increase the immunoglobulin of blood serum(IgG and IgM), and accelerate production of the hemolysin antibody and the proliferation of T lymphocytes.

CONCLUSIONSXue Hanjing oral can reinforce immunological function in mice.

Adjuvants, Immunologic ; pharmacology ; Administration, Oral ; Animals ; Antibody Formation ; Cell Division ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Female ; Hemolysin Proteins ; immunology ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Macrophages, Peritoneal ; physiology ; Male ; Mice ; Organ Size ; drug effects ; Phagocytosis ; drug effects ; Plants, Medicinal ; chemistry ; Poaceae ; chemistry ; Random Allocation ; T-Lymphocytes ; cytology