Abstract:Aim To investigate whether proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates monocyte-endothelial cell adhesion through Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/cyclooxygenase-2 (COX-2) pathway in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were incubated with oxidized low density lipoprotein (ox-LDL), real-time PCR and Western blot assay for detection of PCSK9, TLR4, NF-κB, COX-2 mRNA and protein expression. After treatment with ox-LDL, recombinant PCSK9 protein was incubated or PCSK9 siRNA was transfected into HUVECs. Then the expression of TLR4, NF-κB p65, COX-2 mRNA and protein was detected. After treatment with ox-LDL, human recombinant PCSK9 protein was incubated with TLR4 inhibitor TAK-242 or NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) to detect mRNA and protein expression of NF-κB p65 and COX-2. After COX-2 inhibitor (NS398) and human recombinant PCSK9 protein were treated successively, THP-1 monocytes were added and monocyte-endothelial cell adhesion was detected. Results ox-LDL up-regulates the mRNA and protein expression of PCSK9, TLR4, NF-κB and COX-2 in HUVECs (all P＜0.05). Human recombinant PCSK9 protein up-regulated TLR4, NF-κB, COX-2 mRNA and protein expression on ox-LDL basis (all P＜0.05). PCSK9 siRNA transfection down-regulated the up-regulation of TLR4, NF-κB, COX-2 mRNA and protein expression by ox-LDL (all P＜0.05). TAK-242 inhibits the up-regulation of TLR4 and NF-κB mRNA and protein expression by human recombinant PCSK9 protein (all P＜0.05). PDTC inhibits the up-regulation of NF-κB and COX-2 mRNA and protein expression by human recombinant PCSK9 protein (all P＜0.05). NS398 inhibits monocyte-endothelial cell adhesion induced by human recombinant PCSK9 protein (all P＜0.05). Conclusion PCSK9 regulates monocyte-endothelial cell adhesion via TLR4/NF- κ B/COX-2 pathway.

Abstract:Aim To investigate the relationship between the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway and cyclooxygenase-2 (COX-2) expression in THP-1 monocytes. Methods Human THP-1 monocyte was used as the research cell, and the time-dependent expressions of STAT3 phosphorylation and COX-2 were detected after IL-6 treatment for 0 to 48 hours. THP-1 monocytes were pretreated with 100 μmol/L S3I-201(an specific inhibitor of STAT3 signaling) for 24 hours and then treated with 10 μg/L IL-6 for certain time. THP-1 monocytes were divided into 4 groups:control group, S3I-201 group, IL-6 group and IL-6＋S3I-201 group, then the changes of STAT3 phosphorylation and COX-2 expression were detected. COX-2 mRNA expression was detected by real-time fluorescence quantitative PCR. The levels of STAT3 phosphorylation and COX-2 protein expression were determinated by Western blot. Results IL-6 could obviously induce STAT3 phosphorylation and COX-2 expression via a time-dependent manner in THP-1 monocytes. Phosphorylation level of STAT3 increased after IL-6 stimulation for only 5 minutes (P＜0.001), meanwhile, COX-2 mRNA and protein expressions was significantly upregulated, reaching peak at 1 h and 2 h respectively (P＜0.001). Compared with control group, COX-2 mRNA and protein expressions were both markedly suppressed in S3I-201 group (P＜0.01). Compared with IL-6 group, phosphorylated STAT3 level was downregulated in IL-6＋S3I-201 group (P＜0.001), and COX-2 mRNA expression was also decreased (P＜0.001), with COX-2 protein expression clearly suppressed (P＜0.05). Conclusion IL-6 is capable of activating the STAT3 pathway in THP-1 monocytes, which may play a role in COX-2 expression and further affect the process of atherosclerotic disease.

Abstract:Aim The present study was to investigate the protective effects of oxymatrine （OMT） on regulation of the expression of cytosolic phospholipase A2 （cPLA2）,cyclooxygenase-1 （COX-1）,COX-2 and prostacyclin synthase （PGIS） in isoproterenol-induced rat heart failure. Methods Heart failure was induced in Sprague-Dawley rats by 5 mg/kg isoproterenol （ISO） subcutaneous injection for 7 days.The rats,maintained on a normal diet,were randomly divided into five groups given control,oxymatrine （100 mg/kg） alone,ISO and ISO with oxymatrine （100 mg/kg or 50 mg/kg）.In groups of ISO and ISO with oxymatrine （100 mg/kg or 50mg/kg）,saline or oxymatrine was administered orally for 7 days prior to the ISO administration.ISO （5 mg/kg） was administered subcutaneously for 7 days with saline or oxymatrine.In groups of control and oxymatrine （100 mg/kg） alone,saline was administered subcutaneously for 7 days.Serum brain natriuretic peptide （BNP） level,haemodynamic parameters,histopathological variables and expression of protein levels were analysed. Results Oral administration of OMT significantly inhibited ISO-induced heart failure,as evaluated by serum BNP and haemodynamic parameters,and histological examinations.Coadministration of oxymatrine increased myocardial level of COX-2 and PGIS,and inhibited COX-1 expression in ISO-induced heart failure rat.Whereas the protein level for cPLA2 was markedly increased by ISO,OMT exerted no effects on ISO-induced elevated protein cPLA2 expression.Compared with control group,any indexes in sham rats treated with OMT （100 mg/kg） alone were unaltered （all P＞0.05）. Conclusion Our results suggest that the favourable effects of oxymatrine for management of heart failure are probably achieved by regulation of the COX-1,COX-2 and PGIS expression.

Abstract:Aim To investigate the effect of Tanshinone ⅡA on endothelial cells’ cyclooxygenase-2 （COX-2） expression and the relative mechanisms. Methods Human aortic endothelial cells were incubated in vitro and then co-cultured with angiotension Ⅱ （100 nmol/L）,Tanshinone ⅡA （1 μmmol/L） and no drugs respectively for 10 minutes,1 hour and 2 hours.The expressions of COX-2 mRNA were measured by real time-PCR and COX-2,p38MAPK,p-p38MAPK,NF-κB and p-NF-κB protein levels were measured by Western blot assay. Results AngiotensionⅡ could induce the expression of COX-2 mRNA and protein （P<0.01） as the same as the phosphorylation of p38MAPK and NF-κB （P<0.01）.Incubating human aortic endothelial cells with angiotensionⅡ and Tanshinone ⅡA suppressed the expression of COX-2 （P<0.01） and the phosphorylation of p38MAPK and NF-κB was also decreased （P<0.01）. Conclusion Tanshinone ⅡA could suppress the expression of COX-2 and it may be achieved through inhibiting the phosphorylation of p38MAPK and NF-κB.

Abstract:Aim To investigate gene expression patterns of atherosclerosis (As) induced by intermittent hypoxia. Methods The total RNA were extracted from the aorta of rabbits and then reverse transcribed to make the probe. The probes hybridisate the gene chip from Afymetrixs company, the result signals were scanned by ScanArray HS-038 and analyzed by Microarray Suite Version 5.0. Results There was total 187 genes down-regulated or up-regulated, classified by cell signal transduction, immunology inflammatory response, extracellular matrix degrade, cell apoptosis, smooth muscle cell related, oncogene or tumor suppressor and cell migration. The expression level of cyclooxygenase-2 (COX-2) mRNA in abdominal aorta was significantly associated with the change of abdominal aorta endarteropathy (P<0.05) . Conclusion The process of As induced by intermittent hypoxia is correlated to many genes, gene chip, the high-flux detecting technique can provide the new ideas for the mechanism research of As. COX-2 plays an important role in As.

Abstract:Aim To observe the expression of angiotensinⅡ-induced cyclooxygenase-2 (COX-2) in cultured human umbilical vascular smooth muscle cells and investigate the influence of aspirin and fluvastatin on it. Methods Vascular smooth muscle cells were prepared from human umbilical arteries. The expression of COX-2 were observed in these cells after stimulated with different concentrations of angiotensinⅡ (AngⅡ) for 1 h, or stimulated with AngⅡ after treated with different concentrations of aspirin or fluvastatin or their combination for 1 h. Reverse transcription-polymerase chain reaction (RT-PCR) and cell immunocytochemistry were used for COX-2 mRNA and protein analysis. Results The expression of COX-2 mRNA was markedly increased under the stimulation of AngⅡ in a dose-dependent manner and reached to the peak when the concentration of AngⅡ was 1.0 μmol/L. This effect was inhibited by aspirin and fluvastatin, and the amount of COX-2 mRNA decreased gradually when the drugs dose increased. The amount of COX-2 mRNA was even lower in the drugs combination group than those in monotherapies groups (p<0.05). Results of the COX-2 protein expression with cell immunocytochemistry were coincident with those of mRNA expression with RT-PCR. Conclusions COX-2 can be induced by AngⅡ in cultured human vascular smooth muscle cells, which may play an important role in the progression of atherogenesis. Aspirin and fluvastatin can co-suppress the expression of COX-2, which may bring a novel therapeutic idea for drugs use.

Abstract:Aim To elucidate how advanced glycation end products(AGE) effected cyclooxygenase-2(COX-2) expression in cultured human umbilical vascular endothelial cells (ECV304). Methods ECV304 were cultured in vitro with AGE-human serum albumin(AGE-HSA).The levels of protein COX-2 were measured by Western blot. Results COX-2 expressed little in ECV304,AGE-HSA could induce COX2 expression,and the expression could be blocked by inhibiting the activation of NF-κB. Conclusion AGE-HSA could induce COX-2 expression by activing NF-κB.This pathobiological effect of AGEs might contribute to vascular lesion.

Abstract:Aim To study the effect of atorvastatin on the expression of cyclooxygenase-2(COX-2) and other proinflammatory molecules in a rabbit model of atherosclerosis and to further explore the potential mechanism of atorvastatin in anti-atherosclerostic inflammation beyond the lowing lipid. Methods Twenty-four male New Zealand rabbits(3 months old)were fed with normal diet(n=8) and high-cholesterol diet(n=16) respectively for 8 weeks,and then the high-cholesterol diet rabbits were assigned to receive either atorvastatin 2.5 mg/(kg·d)(n=8)or starch(n=8).6 weeks later,aortas were removed under deep anesthetization.The femoral arteries were removed to determine COX-2 mRNA by reverse transcription-polymerase chain reaction and the level of COX-2 and matrix metalloproteinase-9(MMP-9) protein by immunohistochemistry. Atherosclerotic lesions were measured by experienced pathologist under Beihang Pathology Imaging Analysis System in aortas from hypercholesterolemic rabbits.Plasma interleukin-6(IL-6) levels were measured by enzyme linked immunosorbent assay(ELISA). Results Compared with placebo-treated group,atherosclerotic area was reduced in atorvastatin-treated group(43.0%±12.5% vs 83.0%±11.6%,p<0.05).COX-2 mRNA expression in aortas from hypercholesterolemic rabbits were significantly increased compared with those from normal rabbits,and significantly reduced by the treatment of atorvastatin(1.03±0.09 vs 0.57±0.10,p<0.05),and the levels of COX-2 mRNA expression were related to both atherosclerotic area and plasma IL-6 levels(r=0.803 and 0.795,both p<0.05).Expression of COX-2 protein in atherosclerostic plaques were significantly increased and reduced by the treatment of atorvastatin(62.4%±8.5% vs 34.3%±8.8%,p<0.05),and the levels of COX-2 were related to the levels of MMP-9 in atherosclerostic plaques(r=0.815,p<0.05). Conclusion In hypercholesterolemic rabbits,atorvastatin can decrease circulating IL-6 level and MMP-9 level in plaque,and the potential mechanism of atorvastatin in anti-atherosclerostic inflammation may be through the COX2 signal pathway.

Abstract:Aim To observe the effects of treatment with celecoxib, a specific cyclooxygenase-2 inhibitor, together with low-dose aspirin on atherosclerosis progression in an apolipoprotein-E knockout (ApoE -/-) mice model. Methods 8-week-old ApoE -/- mice fed on a western-diet were devided into group aspirin (n=10), group aspirin+celecoxib (n=11), and group placebo (n=8). Aspirin, aspirin+celecoxib and placebo were given respectively by oral gavage. Wildtype mice fed on a normal diet were as normal control group (n=6).At 16 weeks of age, serum total cholesterol (TC) and triglyceride (TG) were determined, atherosclerotic lesions in arotic root were evaluated by oil red O staining. Results A small difference in serum TC and TG levels was found either in group aspirin or in group aspirin+celecoxib compared with group placebo (30.2±5.5 mmol/L and 28.6±6.2 mmol/L vs 37.8±8.1 mmol/L,p<0.05, p<0.05); However, there was no significant difference between the two treatment groups. Oil red O staining showed that mean plaque size in arotic root was significant smaller either in group aspirin+celecoxib or in group aspirin compared with group placebo (p<0.01), and it was much smaller in group aspirin+celecoxib (0.042±0.026 mm2 vs 0.068±0.022 mm2, p<0.05). Conclusions Aspirin can slightly decrease serum level of TC in ApoE -/- mice; Celecoxib, together with low dose aspirin, can reduce the progression of atherosclerosis to a much more extent

Abstract:Aim To investigate the change of cyclooxygenase-2 in hypercholesterolemic rabbits carotid artery injured by balloon and the relationship with intimal hyperplasia. Methods Twenty-five New Zealand male rabbits were studied. They were fed with atherogenic diet. Four weeks later, balloon injury was conducted in right carotid artery respectively. A few rabbits were killed at 6 h, 24 h, 1 W, 2 W and 4 W. Blood samples and right carotid artery were collected in time. Intimal hyperplasia was studied by histological morphology method. The expression of tissue cyclooxygenase-2 mRNA was determined by reverse transcription-polymerase chain reaction. Result Intimal hyperplasia was present at day 7 after balboon injury, became more obvious at day 14 and in progression at day 28. The media area was not changed. The intima/media ratio increased as time went on (0.032±0.004,0.030±0.003, 0.030±0.002, 0.251±0.045, 1.111±0.182 and 1.448±0.216). No change was observed in control group. Cyclooxygenase-2 mRNA expression was trace in the carotid atery of the hypercholesterolemic rabbits without balloon injury (0.5±0.21, 0.5±0.25, 0.6±0.19, 0.6±0.26 and 0.5±0.22). The expression increased significantly after balloon injury as time went on (0.6±0.22, 0.8±0.24, 1.2±0.31, 1.6±0.36 and 1.4±0.32). After balloon injury cyclooxygenase-2 expressed stronger in injury group than in control group. Conclusions Cyclooxygenase-2 expressed stronger in injury group than in control group, which indicated that cyclooxygenase-2 may play an important role in restenosis caused by balloon injury.