| Atherosclerosis (AS) is a chronic inflammatory diease in arteries.Ox-LDL is an important risk factor on inducing of atherosclerosis, andplays a critical role in the initiation and development of AS. Nuclearfactor –κappaB (NF-κB), a redox-sensitive transcription factor, hasalso been indicated to play a critical role in both initiaion andexpansion of AS. The activation of NF-κB may be linked to the onsetof atherosclerosis. Apoptosis of foam cells exist in atheroscleroticplaque. Apoptosis is the pathogenesis of AS and destabilization of theatherosclerotic plaque. ox-LDL,cells apoptosis and NF-κB all haverelationship to AS.we devise this experiment to investigation theralationship of them.We incubate U937 cells with ox-LDL to induce cells apoptosis. Thento detect the expression of caspase-3,-8,-9 and NF-κB protein. We wantto investigation the problems followed: (1) The mechanism of U937 cellsapoptosis induced by ox-LDL. (2) The effection of NF-κB on U937 cellsapoptosis induced by ox-LDL. (3) The effection of ACEI on U937 cellsapoptosis induced by ox-LDL. (4) The effection of ACEI on the expressionof NF-κB protein. This will be a new direction on investigation ofinitiation and development of atherosclerosis.Method:U937 cells are incubated and treated with differentconcentrations(0 to 300ug/ml) of ox-LDL for 24 hours,and with 100ug/mlox-LDL for different time (0-72h). we checked the proportion of cellsapoptosis . Then U937 cell3 were preincubated with inhibitor andmedicine. We observe the change of the proportion of cells apoptosis,caspase3,8,9 and NF-κB protein.The degree of U937 cell apoptosis wasdetermined by flow cytometry . The expression of caspase3,8,9 and NF-κBprotein was determined by immune histochemsitry and Western Blot.Result:1. Ox-LDL induced apoptosis in U937 cells in a concentration-andtime-dependent manner. Determined by flow cytometry , we found thatnative LDL almost had no apoptotic evidence. But ox-LDL treatment,especially at 100ug/ml concentration for 24 hours, induced an increaseof apoptosis.2. The effect of the two apoptosis pathway, involved in caspase, onox-LDL induced apoptosis in U937 cells . Ox-LDL treatment, at 100ug/mlconcentration for 24 hours, there were a significant activated caspase-3,-8, -9. And appear the apoptotic optical feature with immunehistochemsitry.3. The effect of the NF-κB on ox-LDL induced apoptosis in U937 cells.Ox-LDL treatment, at 100ug/ml concentration for 24 hours, there werea significant activated NF-κB protein determined by immunehistochemsitry. Pretreatment U937 cells with NF-κB inhibitor PDTC 1hour before with ox-LDL inhibited cell apoptosis and the expression ofcaspase-3, -8, -9. So NF-κB promoted ox-LDL induced apoptosis in U937cells and the apoptosis pathway. Ox-LDL induced apoptosis in U937 cellsthough activating NF-κB.4. The effect of the captopril on ox-LDL induced apoptosis in U937 cells.Pretreatment U937 cells with captopril 1 hour before with ox-LDLinhibited cell apoptosis and the expression of caspase-3, -8, -9 andNF-κB. So captopril inhibited ox-LDL induced apoptosis in U937 cellsinhibiting the activation of NF-κB. (P<0.05)Discussion: Oxidized low-density lipoproteins (ox-LDL) play a critical role inpathogenesis of the atherosclerotic plaque, myocardial ischemia /reperfusion injury, and acute myocardial infarction. Ox-LDL aregenerally thought to contribute to the development and propagation ofatherogensis. Ox-LDL can induce macrophages apoptosis, promoting theformation of foam cell leading to the development of AS. Ox-LDL canstimulate U937 cell differentiate into monocytic-derived macrophages.Ox-LDL induced apoptosis in U937 cells in a concentration-andtime-dependent manner. Ox-LDL treatment, especially at 100ug/mlconcentration for 24 hours, induced an increase of apoptosis. Apoptosis, also called programmed cell death, is an importantprocess of many pathological conditions including atherosclerosis.Ox-LDL can induce apoptosis in a variety of tissues and cells includingendothelial cells, smooth muscle cells and macrophages. The initiationand regulation of apoptosis is highly controlled by a family ofproteolytic enzymes, the caspase. The activity of different caspase iscontrolled by specific apoptosis protein. The caspases of theox-LDL-induced apoptosis invovles two pathways of apical caspaseactivation: 1.Death receptor-mediated caspase-8 pathway: ActivatingFas by the binding of Fas ligand activating a cascade of cysteineaspartate-specific proteases, the caspases (especially, caspase-8 andcaspase-3) that result apoptosis. 2.Mitochondria-mediated caspase-9pathway: the motochondrial release of cytochrome c binding to Apf-1stimulates caspase-9 activity, then resulted in the activating ofcaspase-3, at last leading to apoptosis. Two pathways converge in theactivation of executing caspases including caspase-3. In our experiment,Ox-LDL treatment, at 100ug/ml concentration for 24 hours, there werea significant activated caspase-3, -8, -9 by immune histochemsitry andWestern Blot. The results indicate that the ox-LDL-induced apoptosisinvolved both death receptor and mitochodria pathway. Nuclear factor –κappaB (NF-κB), a redox-sensitive transcriptionfactor, has also been indicated to play a critical role in both initiaionand expansion of AS. The activation of NF-κB may be linked to the onsetof atherosclerosis. NF-κB activates of target genes relevant to thepathophysiology of vessel wall, including cytokines, leukocyte adhesionmolecules, as well as genes that mediate cell survival. Oxidative stress,angiotensin Ⅱ,and so on, are related to NF-κB activation inatherosclerosis. The NF-κB system may coordinate the activation ofmultiple inflammatory genes, also induce genes whose products havesurvival and protective functions that are important in regulating theexpansion and progression of atherosclerotic lesions. The results ofour experiment demonstrate that NF-κB promte ox-LDL induced apoptosisin U937 cells. It indicates that ox-LDL induced apoptosis in U937 cellsthough activating NF-κB.
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