Accelerated Atherosclerosis Effect Of Organophosphate Insecticide And Potential Mechanism

Objective:To explore the effect of chlorpyrifos on the formation of atherosclerosis induced by the high fat diet in New Zealand rabbits and analyze the possible mechanisms.Methods:1.Thirty two healthy male New Zealand rabbits were divided randomly into four groups with eight rabbits in each group:control group, high-fat diet group, chlorpyrifos group and high-fat diet+chlorpyrifos group. Chlorpyrifos (20 mg/kg/d, the LD50 of chlorpyrifos by mouth in rabbit is 1000-2000mg/kg) was administered by lavage every day for six months.2.The levels of serum fat and activities of cholinesterase (CHE), paraoxonase 1(PON1) and alanine aminotransferase were measured respectively. Serum creatinine and blood urea nitrogen were measured respectively..The levels of serum superoxide dismutase (SOD) and malondialdehyde (MDA) were measured respectively.3.The vascular functions test was performed by using isolated blood vessel method.4.The peritoneal macrophages were assembled.and cellular cholesterol efflux was analyzed.5.Area of atherosclerosis plaque of thoracic aorta was measured by SudanⅣ. Common carotid artery and thoracic aorta were fixed in formalin, sliced and HE dyed and pathology analysis system was used.6.The expressions of ABCA1 and PON1 were detected by Real-time PCR and Western blot.Results:1.Serum activities of CHE and PON1 compared with control group were singificantly decreased, but there were not symptom of intoxation and injury of function of liver and kidney, and the level of HDL and SOD were markedly decreased, the level of MDA was increased, PON1 expression of liver was decreased, endothelium-depent and non-dependent relaxation of abdominal aorta was decreased, and expression of ABCA1 in liver, aorta and peritoneal macrophages and cholesterol efflux from peritoneal macrophages were markedly decreased in chlorpyrifos group compared with control group.2.Compared with control group, serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and triglyceride (TG) were singificantly increased, endothelium-depent and non-dependent relaxation of abdominal aorta was decreased, serum activity of PON1 and expression of ABCA1 in liver were singificantly, expression of ABCA1 in liver, aorta and peritoneal macrophages was markedly increased and cholesterol efflux was significantly increased in peritoneal macrophages in high-fat diet group. There was obvious atherosclerosis lesion in thoracic aorta and common carotid artery in high-fat diet group.3.Compared with high-fat diet group, serum activities of CHE and PON1 were singificantly decreased, but there were not symptom of intoxation and injury of function of liver and kidney, the level of SOD was markedly decreased, the level of MDA was increased, endothelium-depent and non-dependent relaxation of abdominal aorta was decreased, PON1 expression of liver was decreased and expression of ABCA1 and cholesterol efflux were singificantly decreased, the atherosclerosis lesion area in thoracic aorta and common carotid artery was increased in high-fat diet+chlorpyrifos group.Conclusion:Subtoxic dose of chlorpyrifos may accelerate formation of atherosclerosis induced by the high fat diet in New Zealand rabbits, which the mechanism may be related to the injury of endothelium, the decrease of ABCA1 expression and cholesterol efflux, the decrease of ABCA1 expression and increase of oxidative stress induced by chlorpyrifos.Objective:To explore the effect of paraoxon (PXN) on the formation of foam cells induced by the oxidized low density lipoprotein (Ox-LDL) and analyze the possible mechanisms.Methods:1.RAW 264.7 macrophages were divided into five groups:control group, foam cells model induced by Ox-LDL group, Ox-LDL+paraoxon (1μmol/l) group, Ox-LDL+paraoxon (10μmol/l) group, Ox-LDL+ paraoxon (100μmol/l) group. Cells were maintained at 37℃in 50% CO2-95% air in an incubator for 72 h.2.The cellular lipid accumulation was examined by oil red staining. The cellular contents of total cholesterol (TC) and free cholesterol (FC) were deteeted by high performance liquid chromatography assays. Cholesterol efflux from macrophages was examined.3.The expressions of CD36, ATP-binding Cassette Transporter A1 (ABCA1) and cholesterol acyltransferases-1 (ACAT-1) were detected by Real-time PCR and Western blot.Results:1.Oil red-staining positive cells were found and macrophages were filled with lipid droplet in foam cells model group. Compared with the foam cells, paraoxon (10μmol/l,100μmol/l) significantly increased the amount of oil red-staining positive cells and the contents of lipid droplet of foam cells. There was no significant differences between paraoxon (1μmol/l) group and control group.2.Compared with the control macrophages group, the contents of TC, FC, eholesterylester (CE) and CE/TC ratio in model group were significantly increased in foam cells model group. Paraoxon (10μmol/l, 100μmol/l) significantly increased the contents of TC, FC, eholesterylester (CE) and CE/TC ratio compared with foam cells model group. There was no significant differences between paraoxon (1μmol/l) group and control group.3. Compared with the control macrophages group, cholesterol efflux was markly increased in foam cell model group. Compared with the foam cell model group, cholesterol efflux was significant increased in paraoxon (10μmol/l,100μmol/l) group. Paraoxon (1μmol/l) did not influence cholesterol efflux.4. Expression of CD36 was markly up regulated in foam cells compared to the control cells. Compared with the foam cell, expression of CD36 was significantly increased in paraoxon (10μmol/l,100μmol/l) group. Paraoxon (1μmol/l) did not affect the expression of CD36.5. Compared with the control macrophages group, expression of ABCA1 was markly up regulated in foam cells group. Paraoxon (10μmol/l,100μmol/l) significantly down regulated the expression of ABCA1 compared to foam cells group. There was no significant differences between paraoxon (1μmol/l) group and control group.6. Expression of ACAT1 was markly increased in foam cells compared to the control cells. Compared with the foam cell, expression of ACAT1 was significantly up regulated in paraoxon (10μmol/l,100μmol/l) group. Paraoxon (1μmol/l) did not affect the expression of CD3Conclusion:Paraoxon accelerates the formation of foam cell induced by Ox-LDL, which the mechanism is related with up-regulation of expression CD36 and ACAT1 and down-regulation of expression ABCA1 induced by paraoxon.Objective:To investigated the effect of paraoxon on ABCA1 expression and ABCA1-dependent cholesterol efflux, and then examined the role of cyclic adenosine monophosphate (cAMP) signaling pathway in the regulation of ABCA1 expression and ABCA1-mediated cholesterol efflux by paraoxon in RAW 264.7 macrophage-derived foam cells.Methods:1. RAW 264.7 macrophages were cultured in the medium with 50μg/ml Ox-LDL to induce foam cells. Foam cells were divided into several groups:control group, paraoxon group:paraoxon (1,10 and 100μmol/1) for 24 h and paraoxon (100μmol/1) for 6,12,24 h.2. The cellular contents of total cholesterol (TC) and free cholesterol (FC) were deteeted by high performance liquid chromatography assays. Cholesterol efflux from macrophages was examined.3. The level of intracellular cAMP was measure by ELISA. The activities of adenylate cyclase(AC) and phosphodiesterase (PDE) were examined.4.The expressions of ATP-binding Cassette Transporter A1 (ABCA1) was detected by Real-time PCR and Western blot.Results:1.Paraoxn significantly down regulated ABCA1 expression and reduced ABCA1-dependent cholesterol efflux and increased the levels of the total, free and esterified cholesterols in a time-and dose-dependent manner.2.Paraoxon also markedly reduced cAMP level and decreased adenylate cyclase (AC) activity and increased cAMP-specific phosphodiesterase (PDE) activity.3.CAMP analogs dibutyryl cyclic adenosine monophosphate (dBcAMP) markedly compensated the down-regulation of ABCA1 expression and partly compensated the reduction of ABCA1-mediated cholesterol efflux induced by paraoxon.4.Both adenylate cyclase agonist forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) markedly compensated the suppression effect on cAMP level induced by paraoxon.Conclusion:Paraoxon down regulates ABCA1 expression and decreases ABCA1-mediated cholesterol efflux through cyclic AMP signaling pathway in RAW 264.7 macrophage-derived foam cells.Objective:To investigate the effect of ectogenic PON1 transfected on accelerated atherosclerosis effect of paraoxon in RAW 264.7 macrophage-derived foam cellsMethods:1. Experiments were divided into several groups:RAW 264.7 macrophages group; Ox-LDL+RAW 264.7 macrophages group; paraoxon (100μmol/1)+Ox-LDL+RAW 264.7 macrophages group; human PON1 gene transfection+paraoxon (100μmol/l)+Ox-LDL+ RAW 264.7 macrophages group. The plasmid DNA (human PON1 gene in pcDNA3.1+plasmid) was introduced into the RAW 264.7 macrophages. Foam cells were induced by 50μg/ml Ox-LDL and 100μmol/1 paraoxon was treated for 48 h.2. The cellular lipid accumulation was examined by oil red staining. The cellular contents of total cholesterol (TC) and free cholesterol (FC) were deteeted by high performance liquid chromatography assays. Cholesterol efflux from macrophages was examined.Results:1. The contents of TC, FC, eholesterylester (CE) and CE/TC ratio were significantly increased and cholesterol efflux was markly decreased in foam cells group compared with RAW macrophages group. The contents of TC, FC, eholesterylester (CE) and CE/TC ratio were significantly increased and cholesterol efflux was markly decreased in foam cells treated with paraoxon group compared with foam cells group. Compared with foam cells treated with paraoxon, the contents of TC, FC, eholesterylester (CE) and CE/TC ratio were significantly decreased and cholesterol efflux was markly increased in ectogenic PON1 transfected group.2. Cholesterol efflux was markly increased in foam cells group compared with RAW macrophages group. Cholesterol efflux was markly increased in foam cells treated with paraoxon group compared with foam cells group. Compared with foam cells treated with paraoxon, cholesterol efflux was markly increased in ectogenic PON1 transfected group.Conclusion:Ectogenic PON1 inhibits accelerated atherosclerosis effect of paraoxon in RAW 264.7 macrophage-derived foam cells.