Study On The Mechanism Of Oxidized LDL Induced U937 Cell Apoptosis And The Effect Of LOX-1 And Captopril

Ox-LDL is an important risk factor on inducing ofatherosclerosis, and plays a critical role in the development of foamcells. Macropgage cell swallow ox-LDL, one side resulting in theaccumulation of cholesterol then cell apoptosis to form foam cells,on the other side resulting in mononuclear cells adhesion andchemoattractant to endothelial cells then differentiation tomacrophage cells. Whereas ox-LDL can induce the differentiation ofU937 cells to macrophage cells, then cell apoptosis to form foam cells.This will be a new direction on investigation of accruing anddevelopment of atherosclerosis.Method:U937 cell 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 apoptosis and theexpression of LOX-1 . Then U937 cell were preincubated with differentinhibitor and medicine. We observe the change of apoptosis,proteolyticenzymes and LOX-1.The degree of U937 cell apoptosis was deterimed byflow cytometry . The expression of LOX-1 was determined by cell ELISAand Western Bblot. The expression of caspase3,8,9 is detected by immunehistochemsitry and Western Blot.Result:1. Ox-LDL increased the U937 cell apoptosis and the expression of LOX-1in a concentration and time dependent manner, and the maximalexpression at 24 hours, 100ug/ml. Native LDL had no effect on theexpression of LOX-1 and apoptosis.2. Incubation U937 cell with ox-LDL for 24 hours can significantupregulate the expression of caspase3, 8, 9, and appear the apoptoticoptical feature with immune histochemsitry. After incubation of 48hours the number of cells significant decresed, the cell fragmentincreased.3. Preincubation U937 cell with inhibitor of LOX-1 PIA, carrageenanand captopril for 1 hour before with ox-LDL significantly suppressedthe cell apoptosis,and restrained the increase of LOX-1 and caspase3,8,9 (P<0.05).4. Preincubation U937 cell with inhibitor NF-κB PDTC the expressionof LOX-1 decreased compared with ox-LDL group.Discussion:Ox-LDL can induce macrophages apoptosis then ncrosis, promotingthe formation of foam cell leading to the development of AS. Ox-LDLcan stimulate U937 cell differentiate into monocytic-derivedmacrophages. ox-LDL increased the U937 cell apoptosis in aconcentration and time dependent manner, and the maximal expressionat 24 hours, 100ug/ml. After incubation of 48 hours the number of cellssignificant decresed, the cell fragment increased leading to necrosis.It is sequent event after apoptosis leading to foam cell formation.Cells internalize and degrade ox-LDL througy a putativereceptor-mediated pathway. The lectin-like oxidized LDL receptor-1(LOX-1) is a new receptor different from scavenger receptor. It hasaltitudinal ligand specify and can support binding, internalizationand proteolytic degradation of ox-LDL, but not native LDL and ac-LDL.It means that the ox-LDL binding to LOX-1 is special. In our experimentox-LDL can induce the expression of LOX-1 on U937 cell. Ox-LDL bindsto LOX-1 rusulted in U937 cell apoptosis. This is not reported in-andout-country. Human monocytes do not expressed LOX-1, but when itdifferentiates into macrophgage, there is LOX-1 expressed. LOX-1played a role on uptaking ox-LDL at the early of monocytedifferentiating into macrophages.Apoptosis also called programmed cell death. Steps leading toox-LDL-induced apoptosis remain unclear. The initiation andregulation of apoptosis is highly controlled by a family of proteolyticenzymes. The caspases of the ox-LDL-induced apoptosis invovles twopathways of apical caspase activation: 1.Death receptor-mediatedcaspase8 pathway: Activating Fas by the binding of Fas ligandactivating a cascade of cysteine aspartate-specific proteases, thecaspases (especially, caspase8 and caspase3) that result apoptosis.2.Mitochondria-mediated caspase9 pathway: the motochondrial releaseof cytochrome c binding to Apf-1 stimulates caspase9 activity, thenresulted in the activating of caspase3, at last leading to apoptosis.Two pathways converge in the activation of executing caspasesincluding caspase3. In our experiment, we tese the casapse9, 8 and 3,finding that incubation U937 cell with ox-LDL can upregulate theexpression of caspase3, 8, 9. The way that ox-LDL mediated apoptosis inU937 cells is unclear. Study testified that Cu-oxLDL-mediated apoptosis in U937cells involved exclusively the mitochondrial pathway, whereasHOCl-oxLDL-induced apoptosis in monocytic U937 cells involves the twopathways of apical caspase activation. But the study explained it only throughinhibitor of caspase protein. We checked the expression of caspase3, 8, 9 byimmune histochemsitry and Western Blot. The results indicate that theox-LDL-induced apoptosis involved both death receptor and mitochodriapathway. Preincubation U937cell with inhibitor of LOX-1 the apoptosisand caspases decrease. The result suggests that ox-LDL can induce U937cell apoptsis through activating the LOX-1, and the ox-LDL-inducedapoptosis involved both death receptor and mitochodria pathway.The relation between LOX-1 and NF-κB is still unclear. In fact,the 5'-flanking region of the LOX-1 gene contain a consensus NF-ΚB binding site-like sequence, suggesting that transcriptionalregulation mediated by NF-ΚB might be involved in LOX-1 gene. NF-ΚB may participate the ox-LDL mediated the expression of LOX-1. Atthe same time, ox-LDL binding to LOX-1 can promote the activation ofNF-ΚB. Therefore LOX-1 and NF-ΚB may influence each other. It shouldbe tested furthermore.AngII can promote the development of atherosclerosis via AT-1, andcan induced LOX-1 mRNA and protein expression and ox-LDL uptake. ACEinhibitor can restrain the conversion from AngI to AngII, and lowerthe LOX-1, and suppressed the uptake of ox-LDL to slower thedevelopment of atherosclerosi. In vitro the experiment has tested theantioxidized function of ACEI, it may relate to the –SH. Our researchindicated ACEI, captoril can protective U937 cell from ox-LDLinduced-apoptosis via suppressing the expression of LOX-1 and...