| Reactive oxygen species (ROS) are a group of oxygen containing chemicals produced in vivo, which have high reactivity with other molecules. At physiological condition, generation and scavenging of ROS maintains balanced kinetically. Once, the generation is over the capability of scavenging, oxidative stress occurs. Excess ROS can induce not only lipid peroxidation and glycoxidation, but also protein oxidation. Biochemical and structural modifications of proteins induced by oxidative attack may lead to functional alterations and in particular to the progressive loss of their metabolic, enzymatic or immunologic properties. Oxidation of amino acid residues such as tyrosine may lead to the formation of dityrosine, protein aggregation, cross-linking, and fragmentation. Advanced oxidative protein products (AOPP) were one of recently found oxidized protein products. AOPP were first found by Witko-Sarsat et al in the plasma of uremia patients in 1996. They were characterized as having specific absorbance at 340 nm in acidic condition and containing massive carbonyls and dityrosines. They were mainly present in two distinct peaks at 600 and below 80KD in uremic plasma by size-exclusion chromatgraphy. The portion of 600KD was found to be the aggregates of albumin by protein electrophoresis. Currently AOPP levels have been found to be elevated significantly in mild and moderate CRF patients and increased with the deteriorating of renal function andreached the highest levels at the stage of hemodialysis. The finding shows that there is remarkable oxidative stress in the mild stage of uremia.AOPP may be produced by oxidation of albumin by HOC1 formed through myeloperoxidase (MPO)-catalyzing reaction between chloride and H2O2 in circulating neutrophil in vivo. Oxidation of albumin by HOC1 in vitro could get AOPP which is similar to that in plasma in CRF patients. A close relationship between AOPP levels and specific monocyte activation markers such as neopterin, tumour necrosis factor (TNF-a) and its soluble receptor has been observed in CRF patients. AOPP have been found to be capable of inducing respiratory burst of neutrophil and monocyte in vitro, which suggested that AOPP might be inflammatory uremic toxin. It has been demonstrated that AOPP contain massive active carbonyls. The carbonyls can cause a series of biological effects such as inducing the secretion of cytokines, cell adhesion molecules, growth factors and so forth. Whether AOPP have the same function as carbonyls is not clear yet.The aim of present study was to investigate effect of advanced oxidation protein products (AOPP) on the secretion of TNF-a in human monocytes and its mechanismMethodsHuman monocyte line THP-1 and healthy subject peripheral blood monocytes (PBMC) were used as target cells to investigate the effect of advanced oxidation protein products (AOPP) on the secretion of TNF-a in human monocytes and its mechanism1. Preparation of endotoxin free AOPP-BSA in vitro AOPP-BSA were prepared according to the method described byWitko-Sarsat et al. Briefly, endotoxin free BSA were incubated with HOCl at molar ratio of 1/70, 1/140 and 1/280 at room temperature for 30 min and then dialyzed against PBS to remove any free HOCl in the solution and stored at 4C after sterilized by passing through a 0.22 u membrane. AOPP were determined by measuring absorbance at 340 nm in acidic condition and were calibrated with chloramines-T in the presence of potassium iodide. 2. Isolation of monocytes from healthy human peripheral bloodMonocytes from healthy human peripheral blood were isolated by the method of gradation density centrifugation. The anticoagulant whole blood were mixed with 6% Dextron at volume ratio 5:1 and kept for 60 min at 37 C. Then upper plasma layer were collected and put on the layer of Ficoll solution (relative density 1.077) and centrifuged at 500 g for 20 min. The nebulous portion in the middle containing monocytes were collected and washed with D-Hank's solution. Cells were incubated in 5% CO2 at 37C for 2 hours, and then washe... |
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