Dissertation > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Renal failure

Inflammatory Cytokines Production of Human Monocyte Stimulated by Advanced Glycation End Products: Signal Transduction

Author LiuYang
Tutor HouFanFan;LiuShangXi
School First Military Medical University
Course Internal Medicine
Keywords advanced glycation end product ROS(superoxide anion radical) NADPH oxidase Reaction oxigon specious (ROS) Interleukin-1β(IL-1β) Tumor necrosis factor-α (TNF-α) monocyte
CLC R692.5
Type Master's thesis
Year 2004
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Cardiovascular disease (CVD)is one of the most important complications in patients with chronic renal failure (CRF), and is also a leading cause of mortality in end-stage renal disease (ESRD) and receiving regular hemodialysis. The incidence of CVD in CRF patients is 10-20 times higher than that of non-CRF patients with similar ages, therefore, the CVD complication is also called "accelerated atherosclerosis" . The pathogenesis of the CVD is still unknown. Our previous studies reveal that serum advanced glycation end products (AGE) level is increased in CRF patients, and AGE precipitate is also demonstrated in arterial tissues of those patients, which implies that excessive AGE accumulation may be involved in the pathogenesis of CVD in CRF patients, and thereby AGE is regarded as a new "pathogen" for atherosclerosis.AGE are heterogeneous compounds of the end products of nonenzymatical glycation and oxidation of proteins and lipids. Their accumulation in vascular tissues may result in arterial sclerosis. AGE are dependent on binding cellsurface receptors to produce pathophysiological responces. Receptors for advanced glycation end products (RAGE) are specific receptors recognizing AGE and have been attributed to mediate the different biological effects in different cell types. Interaction of RAGE and AGE HSA been shown to induce monocyte-macrophage chemotaxis, and increase the production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which further results in the increased metastasis of inflammatory cells by upregulating the endothelial expression of adhesion molecules. Thus the inflammation is exacerbated, and subsequently atherosclerosis accelerated. Recent data demonstrate that RAGE expression is much higher in monocytes from CRF patients than from non-CRF patients. The increased RAGE level may augment the monocyte responses to AGE, subsequently resulting in increased serum levels of IL-1β and TNF-α in CRF patients. Besides, in vitro experiments show that TNF-α is able to increase RAGE expression in cultured monocytes, thus forms a vicious circle, which may account for the higher incidence of atherosclerosis in CRF patients than in non-CRF ones.AGE induced secretion of IL-1β and TNF-α by monocytes is involved in the pathogenesis of CVD in CRF patients. Little is known about the mechanism(s) mediating the increased production the cytokines by AGEs. In the presentstudy, we have examined the effects of AGE and RAGE interaction on inflammatory factors secretion by human monocytes, and signal transduction pathways mediating the effects were also explored.Methods Human monocyte were isolated from healthy adultvolunteers. The following experiments were conducted to investigate the effects of AGEs-modif ied protein on IL?β and TNF-α production by the cells.1. Culture of human monocytes.Human monocytes were prepared by density gradient separation of peripheral blood from healthy volunteers, and incubated in RPMI1640 medium containing 10% (v/v) fetal bovine serum (FBS) at 37C under 5% C02 in air, followed by a wash two hours later to remove the nonsubstrate-αttached cells. Then the attached cells were trypsinized and diluted to a certain concentration for further culture and treatment.2. Analysis of IL?β and TNF-α secreted by monocyte.Monocytes were treated with 50 u g/mL of AGE-HSA or HSA control, and then the supernatants were subjected to enzyme-linked immunoadsorbent assay (ELISA) for determination of IL-1β and TNF- a levels. Next, monocytes were pretreated with anti-RAGE IgG at various doses, apocynin and SB 203580 for 1hr, respectively, before theaddition of AGE-HSA 50 ug/mL, and allowed to stand for 24 hr. The culture medium IL-1β and TNF- a levels were measured as detailed above.3. Analysis of 02- produced by monocyteMonocytes were treated as above. The medium were collected and added the specific 02- probe, 7-dihydroimidazo[l,2-α]pyrazine-3-one (MCLA), and then subjected to chemiluminescence analysis for determination of 02- level

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