ABSTRACT
Cerebral ischemia/reperfusion injury is partially mediated by thrombin, which causes brain damage through protease-activated receptor 1 (PAR1). However, the role and mechanisms underlying the effects of PAR1 activation require further elucidation. Therefore, the present study investigated the effects of the PAR1 antagonist SCH79797 in a rabbit model of global cerebral ischemia induced by cardiac arrest. SCH79797 was intravenously administered 10 minutes after the model was established. Forty-eight hours later, compared with those administered saline, rabbits receiving SCH79797 showed markedly decreased neuronal damage as assessed by serum neuron specific enolase levels and less neurological dysfunction as determined using cerebral performance category scores. Additionally, in the hippocampus, cell apoptosis, polymorphonuclear cell infiltration, and c-Jun levels were decreased, whereas extracellular signal-regulated kinase phosphorylation levels were increased. All of these changes were inhibited by the intravenous administration of the phosphoinositide 3-kinase/Akt pathway inhibitor LY29004 (3 mg/kg) 10 minutes before the SCH79797 intervention. These findings suggest that SCH79797 mitigates brain injury via anti-inflammatory and anti-apoptotic effects, possibly by modulating the extracellular signal-regulated kinase, c-Jun N-terminal kinase/c-Jun and phosphoinositide 3-kinase/Akt pathways.
ABSTRACT
AIM: To form soluble HLA-G1-peptide complex by refolding in vitro, and to study its immune function. METHODS: The heavy chain and beta(2m) of sHLA-G1 were expressed as insoluble aggregates in E. coli, and then the two subunits were refolded to form HLA-G1-peptide complex by dilution method in the presence of specific peptide. The refolded product was purified through Sephadex G-75 gel filtration. The purified product was identified by Western blot with mAb W6/32. The function of soluble HLA-G1 was explored from following three aspects, namely, the influences on cytotoxicity of NK cells, on proliferation of T cells in mixed lymphocyte culture and apoptosis of activated T cells. RESULTS: The refolded complex was recognized by mAb W6/32. It effectively inhibited cytotoxicity of NK cells and proliferation of T cells, and induced apoptosis of activated T cells. CONCLUSION: The refolding of soluble HLA-G1-peptide complex has been successfully realized in vitro. The complex can inhibit the functions of NK cells and T cells.
Subject(s)
HLA Antigens/immunology , HLA Antigens/metabolism , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Renaturation , Animals , Apoptosis/immunology , Blotting, Western , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , HLA Antigens/chemistry , HLA-G Antigens , Histocompatibility Antigens Class I/chemistry , Humans , Killer Cells, Natural/immunology , Protein Folding/drug effects , Protein Renaturation/drug effects , Urea/pharmacologyABSTRACT
AIM: To induce and expand dendritic cells (DC) from rat bone marrow in vitro and identify their biological characterization. METHODS: The rat bone marrow cells were collected and cultured for 48 hours and the floating cells were removed. Then IL-4 and GM-CSF were added into the fresh medium. After 2 weeks, the morphological character of the cultured DCs was observed under light microscope and transmission electron microscope. Expressions of MHC class II molecule, B7-1 and B7-2 were detected by flow cytometry. The cultured DCs were co-cultured with allogenic T cells derived from rat spleen. T cell proliferation was measured by MTT colorimetry. RESULTS: The cultured DCs had the typical morphological characterization of DC, and the expression rates of MHC class II molecule, B7-1 and B7-2 were 74.2%, 81% and 76% respectively. The cultured DCs could notably stimulate the proliferation of allogeneic T cells. CONCLUSION: The adherent culture of rat bone marrow cells, and co-culture with IL-4 and GM-CSF can obtain a number of high purity of DCs, which lay the foundation for study on DC's function.
