通过葡萄糖诱导氧化应激刺激血管内皮细胞中的nadph氧化酶4活性和表达的增加

Increased activity and expression of NADPH oxidase 4 stimulated by glucose induces oxidative stress in vascular endothelial cells Jian LI Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730, China -cell liver obesity -Cell Dysfunction Insulin Resistance FFA VLDL FFA VLDL Glucose Oxidative stress Hypothesis Redox “rheostat“ in cells Where are reactive oxygen species generated? Mitochondrial respirtion-chain enzyme complex xanthine oxidase, NOS, lipoxygenase Phagocyte NADPH oxidase NADPH oxidase of non-phagocytic cells NADPH oxidase is a major source of vascular ROS production. NOX-type NADPH oxidases as superoxide-producing enzymes Fe Fe outside inside I VIIVII III V NH2 H H HH H H NADPH FAD COOH H115 O2 O2- e- Structure of the NADPH oxidase The NOX family of NADPH oxidases gp91phox homology EF-hands Nox1 colon Nox2 phagocytes Nox3 inner ear Nox4 kidney and vascular cells Nox5 testis and lymphoid tissues O2 O2- NADPH e- A B C D DPI:NOX inhibitor; Rotenone: inhibitor of mitochondrial respirtion-chain enzyme complex; L-NAME:NOS inhibitor; Oxypurinol: xanthine oxidase inhibitor NADPH oxidase is a major source of vascular ROS production induced by glucose A: negative control; B: HUVEC; C: 20mM glucose treated HUVEC; D: positive control Glucose induces apoptosis of HUVEC A GC C G C G C:HUVEC; G:20mM glucose-treated HUVEC Western blot Glucose up-regulates the expression of NOX4 -actin NOX4 NOX2 p22phox p47phox p67phox Rac 1 M A N1 N2 N3 N4 p22 p47 p67 Rac1 RT-PCR 0 10 20 30 glucose(mM) A: negative control; B: transfected with GFP-pcDNA3.1; C: transfected with NOX4-pcDNA3.1 Over-expression of NOX4 increases the level of ROS and results in apoptosis A B C A B C A B C A B C NOX4-siRNA decreases the expression of NOX4 and the level ROS in HUVEC C C+G S-FAM S-FAM+G S-NOX4 S-NOX4+G C S-FAM S-NOX4 * * * * * * Mechanism for increased ROS production induced by diabetes and insulin-resistant state Diabetes High glucose level Insulin resistance Obesity Elevated NEFA level De novo DAG synthesis Vascular cells PKC activation NADPH oxidase activation Superoxide ROS Activation of NADPH oxidase Immunoflurescence staining of p47phox PKC/p47phox regulates the activation of NOX4 ROS level Control NaOH DMSO HypericinGlucose PMA ROS level Control Glucose DPI Apocinin Schematic illustration of ROS-mediated NFB activation Glucose activates NFB pathway C G -actin p-NFB NFB IB p-IB VCAM-1 ICAM-1 C G G+DPI NFB DAPI Merge C G G+SB Glucose activates p38MAPK pathway P-p38MAPK p38 MAPK -actin C G G+SB203580 Western blot Hochest/PI staining Immunofluorescence C G G+SB203580 Anti-oxidation agents for the oxidative damage of vascular endothelial cells high glucose level PKC activation PKC inhibitorAngiotensin II NAD(P)H oxidase activation Statins ARB ACEI HDL Probucol Simvastatin decreases the level of MDA and TG in the atherosclerosis model of apo E-/- mice * MDA TCTG SOD * * Simvastatin suppresses the generation of ROS and expression of NOX in atherosclerotic plaque control Simvastatin treatment ROS NOX2 p47PHOX NOX4 Simvastatin has the ability to