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AIM: To observe the effects of dexamethasone and interleukin-10 (IL-10) on the release of proinflammatory cytokines, tumor necrosis factor-?(TNF-?), IL-6 and activation of transcriptional factors, nuclear factor-?B(NF-?B), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) in cultrued human peripheral blood mononuclear cells (hPBMC). METHODS: The hPBMC were divided into control group, lipopolysaccharide (LPS) stimulated group, dexamethasone and IL-10 treated group. The contents of TNF-? and IL-6 in supernatant were mensured by ELISA. The activity of NF-?B, AP-1 and CREB of nuclear abstract were analyzed by electrophoretic morbility shift assay (EMSA). RESULTS: The content of TNF-? was significantly increased 1 hour after LPS stimulation, and it was significantly inhibition by dexamethasone and IL-10. The contents of IL-6 and IL-10 were significantly increased after LPS stimulation for 12 hours. The production of IL-6 was still inhibited by dexamethasone and IL-10, but the production of IL-10 was not affected by dexamethasone. The activities of NF-?B, AP-1 and CREB were significantly increased 1 hour after LPS stimulation. Dexamethasone and IL-10 significantly ihibited their activities, but the effects of dexamethasone was stronger than that of IL-10. CONCLUSIONS: LPS induces the release of several pro and anti- inflammatory cytokines and induces the activation of several transcriptional factors in hPBMC. Dexamethasone and IL-10 can inhibite the production of proinflammatory cytokines TNF-?, IL-6 and the activation of NF-?B, AP-1 and CREB. Dexamthasone has more significant inhibitory effect on AP-1 and CREB than IL-10.
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AIM: To investigate the regulatory effects of lipopolysaccharide binding protein(LBP)on activation of p38 signaling pathway induced by lipopolysaccharide(LPS)in alveolar macrophages. METHODS: The LBP from actue phase rat serum was purified by ammonium sulphate precipitation, Bio-Rex70 resin and the MonoQ column. Rat alveolar macrophages were exposed to LPS (0 01 mg/L or 1 mg/L) the various concentrations of LBP(0 mg/L, 0 01 mg/L, 0 1 mg/L,1 mg/L and 10 mg/L) Western blotting were used to detect phospho-p38 in alveolar macrophages RESULTS: SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and the molecular weight was 60 kD.The binding of lipopolysaccharide to mononuclear cells were enhanced by purified rat LBP. Stimulation of rat alveolar macrophages with LPS at concentration of 0.01 mg/L was LBP dependent. LBP at concentrations up to 1 mg/L was able to increase the activation of p38. However , when LBP concentrations were further increased to 10 mg/L, the phosphorylation levers of p38 were lower as compared with that in the presence of 1 mg/L. Stimulation of rat alveolar macrophages with LPS at concentrations of 1 mg/L was LBP-independent. CONCLUSION: The activation of p38 induced by LPS at lower concentration(0.01 mg/L ) was LBP-dependent, meanwhile, LPS at higher concentration (1 mg/L ) was LBP-independent.
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AIM: The expression of the Fas Antigen and induction of apoptosis by anti-Fas antibody in esoinophils in vitro were investigated. METHODS: Purified eosinophils from health donors were cultured for 72 h in the presence of human IL-5 and with or without anti-Fas monclonal antibody (MoAb) at various concentrations (1-1000 ?g/L). The expression of the Fas antigen on eosinophils was determined by immunocytochemistry. The changes of eosinophils viability and apoptosis were also studied. RESULTS: The Fas antigen was expressed on freshly isolated eosinophils, which had no significant changes after culture in the presence or absence of IL-5. The anti-Fas MoAb at different concentration suppressed significantly the IL-5-mediated eosinophils survival (78%?9%). When eosinophils were cultured in the presence of IL-5 (1?10 4 U/L) with anti-Fas MoAb (1 000 ?g/L), the percentage of alive cell decreased to 30%?12%( P
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Objective To detect the expression of inositol 1,4,5-triphosphate receptor (IP 3R) subtypes in normal rat airway smooth muscle cells (ASMCs) and changes during chronic asthma formation. Methods ASMCs were cultured by collagen enzyme digestion method. The expressions of subtypes of IP 3R were detected by RT-PCR and the purified PCR products were linked with pGEM-T vector for DNA sequencing. Chronic asthma model was established with egg albumin. The changes of IP 3Rs were detected by RT-PCR method. Results All subtypes of IP 3R were expressed in airway smooth muscle cells of normal rats. The expression of IP 3R1 in asthma groups increased obviously as compared with that in the control group (P