OBJECTIVETo investigate the effect of high mobility group box-1 protein (HMGB1) on apoptosis of macrophages in mice.

METHODSThe peritoneal macrophages were obtained from female BALB/c mice. After exposure to different doses of HMGB1 (10, 100, 1000, and 10000 ng/ml) for 24 hours, or exposure to 10 000 ng/ml for different time (6, 12, 24, and 48 hours), cells were stained with Annexin V-PE and 7-amino-actinomycin-D (7-AAD), then determined by flow cytometry and laser scanning confocal microscopy, respectively.

RESULTSExposed to HMGB1 for 24 hours resulted in a dose-dependent induction of apoptosis, with increased levels of apoptosis observed at 100, 1 000, and 10 000 ng/ml compared with controls [(12.91 +/- 3.89)%, (18.76 +/- 3.41)%, and (39.84 +/- 5.98)%, respectively, vs. (4.49 +/- 1.72)%]. Exposed to HMGB1 at 10000 ng/ml for 6, 12, 24, or 48 hours showed a time-dependent increase in apoptosis rate [(18.33 +/- 4.60)%, (25.02 +/- 5.27)%, (38.30 +/- 6.95)%, and (21.81 +/- 7.96)%]. The percentage of apoptotic cells markedly increased with a peak value at 24 hours after HMGB1 stimulation (P<0.01).

CONCLUSIONHMGB1 has a proapoptotic effect on murine peritoneal macrophages.

Animals ; Apoptosis ; Female ; HMGB1 Protein ; physiology ; In Vitro Techniques ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Mice, Inbred BALB C

The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, beta-VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (ILRP) and VLDLR were detected by semi-quantitative RT-PCR. The results demonstrated that VLDL, beta-VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or beta-VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up-regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl-A7-VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride-rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.
Animals ; Arteriosclerosis ; metabolism ; pathology ; Cells, Cultured ; Cholesterol, LDL ; metabolism ; pharmacology ; Female ; Foam Cells ; cytology ; metabolism ; Lipoproteins, VLDL ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Receptors, LDL ; metabolism ; Triglycerides ; metabolism

In this study, we investigated whether retinal soluble proteins, such as interphotoreceptor retinoid-binding protein(IRBP), play a role in the induction of nitric oxide by macrophages in vitro. Cells from the murine macrophage cell line RAW 264.7 and rat and rabbit peritoneal macrophages were incubated in the presence of retinal soluble protein. The nitrite level in the cultured supernatant was evaluated for nitric oxide production using the Griess reaction. IRBP induced significant, dose-dependent nitrite production in both RAW 264.7 and rat peritoneal macrophages. Induction of inducible nitric oxide synthase (iNOS) by retinal proteins was inhibited by the iNOS-specific inhibitor, aminoguanidine, and the tyrosine inhibitor, genistein. These results show that soluble retinal proteins significantly induce nitric acid production by macrophages. Increased production of reactive oxygen species by macrophages in the presence of this soluble retinal protein in vivo may accelerate photoreceptor degeneration in uveitis.
Animal ; Cell Line ; Comparative Study ; Enzyme Inhibitors/pharmacology ; Eye Proteins/pharmacology* ; Guanidines/pharmacology ; Macrophages, Peritoneal/metabolism ; Macrophages, Peritoneal/drug effects* ; Macrophages, Peritoneal/cytology ; Nitric Oxide/biosynthesis* ; Nitric-Oxide Synthase/metabolism ; Nitric-Oxide Synthase/antagonists & inhibitors ; Rabbits ; Rats ; Rats, Inbred Lew ; Retinol-Binding Proteins/pharmacology*

AIMA series of new 1,4-pentadien-3-one derivatives were synthesized to search for new Eight novel hydroxylated non-steroidal anti-inflammatory drugs (NSAIDs) with potent activity.

METHODSE,E-1-(3'-indolyl)-5-( substituted phenyl)-1,4-pentadien-3-one derivatives were synthesized by means of aldol condensation and characterized by 1H NMR, ESI-MS and element analysis. Their anti-inflammatory activity in vitro were evaluated.

RESULTSPreliminary in vitro pharmacological tests showed that all compounds exhibited anti-inflammatory activity.

CONCLUSIONCompounds 4d and 4e exhibited potent anti-inflammatory activity and their anti-inflammatory activity was comparable to resveratrol, and were worthy of further study.

Alkadienes ; chemical synthesis ; pharmacology ; Animals ; Anti-Inflammatory Agents ; chemical synthesis ; pharmacology ; Indoles ; chemical synthesis ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Male ; Mice ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo investigate the influence of high mobility group box-1 protein (HMGB1) on apoptosis of peritoneal macrophages in mice and its receptor mechanism.

METHODSThe peritoneal macrophages were isolated from female BALB/c mice and divided into 4 groups according to different stimuli: i. e, HMGB1 group (with treatment of 10 microg/ml HMGB1 for 24 hours), HMGB1 and anti-receptor advanced glycation end products (RAGE) antibody group (with treatment of 5 microg/ml anti-RAGE antibody for 2 hours followed by HMGB1 stimulation), Recombinant mouse RAGE/Fc chimera (rmRAGE/Fc) and HMGB1 group (10 microg/ml of rmRAGE/Fc and 10 microg/ml HMGB1 were pre-mixed for 2 hours, then the peritoneal macrophages were treated with the mixture), control group (with treatment of phosphate buffer). Expression of RAGE on the surface of macrophages, and the apoptotic rate of the cells were determined by flow cytometry. Laser scanning confocal microscopy was used to observe the apoptosis of the cells.

RESULTSThe percentage of macrophages with positive RAGE expression in HMGB1 group [(54 +/- 12%)] was markedly increased compared to the controls [(13 +/- 5)%, P < 0.01], and fluorescence density of RAGE expression was also significantly different between two groups (126 +/- 10 vs 34 +/- 8, P < 0.01). The occurrence of apoptosis in HMGB1 and rmRAGE/Fc group, as well as in HMGB1 plus anti-RAGE group were much higher than that in control group, and the number of macrophages with apoptosis and necrosis at late stage was obviously increased in HMGB1 group. The apoptic rate in HMGB1 group was (39.5 +/- 2.3)%, which was significantly higher than those in HMGB1 and rmRAGE/Fc group (17.3 +/- 3.6)%, and HMGB1 and anti RAGE group (14.8 +/- 4.8)%, (P < 0.01), which were significantly higher than those in control groups (5.4 +/- 2.3)%, (P < 0.01).

CONCLUSIONRAGE is one of the major receptor to induce apoptosis of macrophages, and the up-regulation of its expression is induced by HMGB1.

Animals ; Apoptosis ; Female ; Gene Expression Regulation ; HMGB1 Protein ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Mice, Inbred BALB C ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism

The effects of hypoxia on the level of reactive oxygen species (ROS), IkappaBalpha tyrosine phosphorylation, transcription of P65 mRNA and NF-kappaB activation in isolated rat peritoneal macrophages were investigated by DCFH-DA fluorescence spectrophotometry, Western blotting and RT-PCR. The results obtained are as follows. (1) During hypoxia, the levels of intracellular ROS began to increase at 1 h, then reached a peak at 2 h, and began to decrease after 3 h. IkappaBalpha tyrosine phosphorylation began to rise after 2 h hypoxia and was the highest after 3 h hypoxia. After 4 h hypoxia it decreased gradually. NF-kappaB activation began to increase after 3 h hypoxia, and reached a peak after 4 h hypoxia. (2) When antioxidant NAC (500 mmol/L) was added into the medium, the level of IkappaBalpha phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 micromol/L), NF-kappaB activation induced by hypoxia was blocked significantly. (3) The expression of p65 mRNA was also elevated markedly during hypoxia. These results suggest that hypoxia may lead to IkappaBalpha phosphorylation and NF-kappaB activation through intracellular ROS, and that the regulation of NF-kappaB activity may involve IkappaBalpha phosphorylation and the expressions of each subunit gene of NF-kappaB.
Animals ; Cell Hypoxia ; Cells, Cultured ; Macrophages, Peritoneal ; cytology ; physiology ; Mice ; NF-kappa B ; biosynthesis ; genetics ; physiology ; Phosphorylation ; RNA, Messenger ; biosynthesis ; genetics ; Reactive Oxygen Species ; analysis ; Signal Transduction

AIMTo study the effect of proanthocyanidins on the COX-2 enzyme activity and COX-2 protein expression in LPS-induced RAW264.7 cells.

METHODSAfter being pretreated with different concentrations of proanthocyanidins for 30 min, and then 1 mg x L(-1) LPS for 9 h, the effect of proanthocyanidins on the activity of COX-2 enzyme in RAW264.7 cells was analysed by radioimmunoassay (RIA). After being pretreated with different concentrations of proanthocyanidins for 30 min, and then 1 mg x L(-1) LPS for 9 h, the effects of proanthocyanidins on the expressions of COX-2 mRNA and protein in RAW264.7 cells were analysed by RT-PCR and Western blotting.

RESULTSThe activity of COX-2 enzyme was not inhibited by proanthocyanidins (0. 8, 4 and 20 mg x L(-1), P > 0.05 vs LPS group), but the activity of COX-2 enzyme was significantly inhibited by 10 micromol x L(-01) NS-398 (P < 0.01 vs LPS group). The expression of COX-2 mRNA was inhibited by proanthocyanidins (0. 8, 4 and 20 mg x L(-1)). The expression of COX-2 protein was inhibited by proanthocyanidins (4 and 20 mg x L(-1)).

CONCLUSIONProanthocyanidins had no effect on the activity of COX-2 enzyme in LPS-induced RAW264. 7 cells. Proanthocyanidins inhibited significantly the expression of COX-2 mRNA and protein in LPS-induced RAW264.7 cells.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Lipopolysaccharides ; Macrophages, Peritoneal ; cytology ; enzymology ; Mice ; Proanthocyanidins ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the potential role of anthocyanins on modulating cholesterol efflux in mouse peritoneal macrophage-derived foam cells and related molecular mechanisms.

METHODSThe macrophages were isolated from pathogen-free NIH mice and were loaded with 50 microg/ml oxLDL for 24 hours, newly formed foam cells were then treated with anthocyanins (cyanidin-3-glucoside, Cy-3-g; or peonidin-3-glucoside, Pn-3-g) at the concentrations of 1 micromol/L, 10 micromol/L, 100 micromol/L for 0 to 36 hours, respectively. The enzymatic-fluorescent method was used to determine cholesterol content in culture medium. ABCA1 expressions at mRNA and protein level were detected by real-time PCR and confocal microscope.

RESULTSCholesterol efflux of macrophage-derived foam cells increased in a time- and dose-dependent manner post anthocyanins treatment. ABCA1 expressions at mRNA and protein levels were also significantly enhanced after anthocyanins treatment in these cells and these effects could be blocked by co-treatment with DIDS, an inhibitor of the transport activities of ABCA1 and blocker of apoAI-mediated cholesterol efflux.

CONCLUSIONThese data demonstrate that anthocyanins induce cholesterol efflux from mouse peritoneal macrophage-derived foam cells via regulating ABCA1 expression.

ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; metabolism ; Animals ; Anthocyanins ; pharmacology ; Cells, Cultured ; Cholesterol ; metabolism ; Foam Cells ; drug effects ; metabolism ; Macrophages, Peritoneal ; cytology ; Mice

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