Influence of atorvastatin on scavenger receptor of monocytes/macrophages in patients with coronary heart disease

AIM: To observe activity of scavenger receptor of macrophage derived from monocytes, the change of inflammation factor in plasma (including CRP, sICAM-1, sVCAM-1) and the influence of atorvastatin on activity of scavenger receptor in patients with coronary heart disease. METHODS: 75 patients with coronary heart disease (CHD), whose plasma lipid levels were normal, were divided into three groups: stable angina pectoris, unstable angina pectoris and acute myocardia infarction. 29 healthy persons were served as control. The level of C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in plasma were determined in all subjects. Monocytes in peripheral blood were dissevered and cultured to transform into macrophage. The influence of atorvastatin on activity of scavenger receptor in macrophage was observed. RESULTS: The level of CRP, sICAM-1, sVCAM-1 and activity of scavenger receptor of macrophage in acute myocardia infarction group were higher than that in stable angina pectoris, unstable angina pectoris and control. Atorvastatin lowered the activity of scavenger receptor of macrophages derived from monocytes in patients with coronary heart disease. Activity of scavenger receptor of macrophages derived from monocytes in patients with coronary heart disease was correlated positively with CRP, sICAM-1 and sVCAM-1. CONCLUSION: Activity of scavenger receptor may be taken as index for monitoring the degree of active vulnerable atherosclerosis plaque. Atorvastatin may inhibit activity of scavenger receptor in macrophages derived from monocytes in patients with coronary heart disease.     

Effects of HDL from patients with coronary artery disease on lipid deposition and apoptosis in mouse peritoneal macrophages

AIM: To compare the effects of high-density lipoprotein (HDL) from healthy subjects (HDL_heathy) and HDL from the patients with coronary artery disease (HDL_CAD) on the lipid deposition and apoptosis in mouse peritoneal macrophages. METHODS: HDL was isolated from healthy subjects, stable CAD patients (HDL_SCAD) and acute myocar-dial infarction patients (HDL_AMI). The accumulation of intracellular lipids was determined by oil red O staining. The apoptosis of macrophages was measured by fluorescence microscopy with annexin-V/PI staining. DCHF-DA, a redox-sensitive dye, was used to detect intracellular reactive oxygen species (ROS) levels. The protein expression of ATP-binding cassette transporter (ABC) A1, ABCG1, Bcl-2 and Bax was determined by Western blot analysis. RESULTS: Lipid deposition in the macrophages was increased significantly after oxidized low-density lipoprotein (ox-LDL) treatment, and the expression of ABCA1 and ABCG1 was up-regulated (P<0.05). Compared with ox-LDL treatment alone, HDL_healthy decreased lipid deposition in the macrophages and up-regulated the expression of ABCA1 and ABCG1 (P<0.05), while treatment with HDL_SCAD or HDL_AMI further decreased lipid deposition in the macrophages and down-regulated the expression of ABCA1 and ABCG1 (P<0.05). Compared with HDL_SCAD treatment, lipid deposition in the macrophages was further increased after HDL_AMI treatment, and the expression of ABCA1 and ABCG1 was down-regulated (P<0.05). HDL_healthy decreased the levels of intracellular ROS and apoptosis by increasing the level of antiapoptotic protein Bcl-2 and reducing the expression of proapoptotic protein Bax. In contrast, HDL_SCAD and HDL_AMI had opposite effects on the intracellular ROS, the cell apoptosis and the expression of apoptosis-related proteins Bcl-2 and Bax. CONCLUSION: HDL_CAD promotes lipid accumulation in macrophages and induces macrophage apoptosis. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in CAD.     

Mast cells change cellular cholesterol content and inhibit cholesterol efflux from THP-1 macrophage-derived foam cells

AIM:Mast cells (MC) are present in the arterial intima,the site of atherogenesis. The present studies explore the effect of MC on cholesterol content,distribution and efflux in THP-1 macrophage-derived foam cells (THP-1FCs）. METHODS:THP-1FCs were incubated with high-density lipoproteins 3 (HDL₃) in the absence or presence of mast cell granules (MCGs) harvested from compound 48/80-stimulated rat peritoneal MC. The intracellular cholesterol level,cholesterol effluxing capacity,ATP-binding cassette transporter A1(ABCA1) mRNA and HDL₃ treated with MCGs were detected to characterize the role of MC on intracellular cholesterol. RESULTS:MCGs had high levels of cellular total cholesterol(TC),free cholesterol(FC) but not esterifed cholesterol(EC) compared to control group where the TC concentrations ranged from 527.3 mg/g to 917.9 mg/g cellular protein with EC accounting for 7.6% of the cholesterol. Cholesterol efflux was 14% less in MCGs group compared to control group. ABCA1 mRNA expression in MCG-treated THP-1FCs remained unchanged in 20 hours. In contrast,treatment of HDL₃ with MCGs resulted in rapid degradation of the main HDL₃ apoliproteins,apoA-Ⅰ. SDS-PAGE revealed that a minor polypeptide band with about 26 kD molecular mass appeared below the apoA-Ⅰband. Densitometric analysis of the gel demonstrated that ≈ 28% of apoA-Ⅰhad been degraded by the MCGs. CONCLUSION:These results indicate that MC decreases cholesterol efflux,increases cellular accumulation in TC and FC by depleting HDL₃ and apoA-Ⅰ,but not by inhibiting ABCA1 mRNA expression.     

The influence of oral simvastatin and vitamine-E on serum LDL oxidation and platelet activation in coronary artery disease patients

AIM: Using simvastatin and vitamine E (Vit-E) treatment to coronary artery disease (CAD) patients with low HDL, to investigate the relationship between Ox-LDL, platelet activation and HDL. METHODS: 40 CAD patients with low HDL were divided into two groups (A and B): A group oral simvastatin, B group oral simvastatin and Vit-E. The level of serum Ox-LDL, TXB₂ and GMP-140 were measured before and after treatment. The relationship between Ox-LDL, TXB₂, GMP-140 and HDL were analysed. RESULTS:The level of serum HDL was significantly increased in A and B group after treatment and attained normal level. The level of serum Ox-LDL, TXB₂ and GMP-140 were decreased significantly after simvastatin and Vit-E treatment and neared normal. CONCLUSIONS:This study confirmed that HDL can effectively refrain LDL oxidation. It also revealed that Vit-E and simvastatin treatment were more effectively refrained platelet activation by increasement of HDL and decreasement of Ox-LDL.     

Effects of oxidized low density lipoprotein and lysophosphatidylcholine on cholesterol efflux from mouse peritoneal macrophage foam cells

AIM:To explore the effects of oxidized low density lipoprotein (OxLDL) and one of its component— lysophosphatidylcholine (LPC) on cholesterol efflux from mouse macrophage foam cells.METHODS:(1) Cholesterol efflux induced by apoAI from mouse peritoneal macrophage foam cells loaded with OxLDL or acylated LDL(AcLDL) was measured. (2) Cholesterol efflux induced by LPC and apoAI from macrophage foam cells separated from normal or apoE gene deficient (E⁰) mouse loaded with AcLDL were measured.RESULTS:(1) When the macrophage foam cells were incubated with apoAI, cholesterol efflux from AcLDL-induced macrophage foam cells increased significantly compared to that of OxLDL-induced macrophage foam cells. (2) LPC promoted cholesterol efflux from macrophage foam cells in relation to both dosage and time. When LPC was incubated with E⁰ mouse macrophage foam cells, the released cholesterol mass was significantly lower than that of normal mouse macrophage foam cells. It was also found that cholesterol efflux induced by apoAI normally occurred in E⁰ mouse macrophage foam cells.CONCLUSIONS:(1) OxLDL accumulated cholesterol in macrophages and impair cholesterol efflux. (2) LPC induced cholesterol efflux from macrophage foam cells, which may occur via apoE pathway.     

Serum CTRP1 and CTRP7 levels are associated with coronary atherosclerotic heart disease and diabetes mellitus

AIM To investigate the relationship between the serum levels of C1q/TNF-related protein (CTRP) 1 and CTRP7 and coronary atherosclerotic heart disease (CAD) in the patients with or without type 2 diabetes mellitus (T2DM). METHODS Totally 138 cases of participants were selected, and divided into control group (without T2DM or CAD; n=40), T2DM group (with T2DM, but without CAD; n=30), CAD group (with CAD, but without T2DM; n=40), and CAD+T2DM group (with both T2DM and CAD; n=28). The serum levels of CTRP1 and CTRP7 in these participants were measured by ELISA. RESULTS (1) Compared with control group, serum CTRP1 level in CAD group and CAD+T2DM group was significantly increased (P<0.05 or P<0.01), and serum CTRP7 level in CAD, T2DM and CAD+T2DM groups was significantly decreased (P<0.05 or P<0.01). (2) Increased serum CTRP1 level was a risk factor for CAD [odds ratio (OR)=1.136; 95% confidence interval (CI): 1.010~1.278; P=0.034). Sex, hypertension and serum triglyceride level were also correlated with CAD. Decreased serum CTRP7 level was a risk factor for T2DM (OR=0.984; 95% CI: 0.969~0.999; P=0.038). Sex, hypertension and serum high-density lipoprotein cholesterol level were also correlated with T2DM. Increased serum CTRP1 level was a risk factor for CAD+T2DM (OR=1.009; 95% CI: 1.005~1.203; P=0.040). Hypertension was also a risk factor for CAD+T2DM. (3) In CAD group, serum CTRP1 level had certain diagnostic value, and the area under the receiver operating characteristic (ROC) curve (AUC) was 0.670 (95% CI: 0.580~0.760; P=0.001), but serum CTRP7 level had no diagnostic value for CAD. In T2DM group, both serum CTRP1 and CTRP7 levels had diagnostic value, and the AUC values of their ROC curves were 0.607 (95% CI: 0.505~0.710; P=0.032) and 0.665 (95% CI: 0.574~0.756; P=0.001), respectively. In CAD+T2DM group, both serum CTRP1 and CTRP7 levels had diagnostic value, and the AUC values of their ROC curves were 0.666 (95% CI: 0.552~0.781; P=0.007) and 0.632 (95% CI 0.517~0.747; P=0.032), respectively. CONCLUSION Increased serum CTRP1 level and decreased serum CTRP7 level are associated with increased risk of T2DM and CAD. Increased serum CTRP1 level is a risk factor for CAD and CAD+T2DM, while decreased serum CTRP7 level is a risk factor for T2DM. Serum CTRP1 level has certain specificity and sensitivity for the diagnosis of CAD, T2DM and CAD+T2DM, while serum level of CTRP7 has certain specificity and sensitivity for the diagnosis of T2DM and CAD+T2DM.     

Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

AIM: To study the protective effect of puerarin on the atherosclerosis of RAW264.7-derived foam cells. METHODS: The model of foam cells was established by incubating the RAW264.7 cells with ox-LDL. The cholesterol uptake was evaluated by a DiI-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264.7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3Ⅱ, P62, CD36, ABCA1, LAL and p-AMPK were determined by Western blot. RESULTS: Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3Ⅱ, ABCA1 and LAL in puerarin group were higher than that in ox-LDL group, while the protein levels of P62 and CD36 were obviously decreased, and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3Ⅱ, ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION: Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264.7 macrophages, which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation, and reduce the uptake of lipids by CD36 negative regulation.     

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

AIM:To investigate the role of apolipoprotein E(ApoE) in cholesterol efflux mediated by ATP-binding cassette transporter A1(ABCA1) and ATP-binding cassette transporter G1(ABCG1). METHODS:RAW 264.7 cells were seeded in either 6-well or 24-well plates, and then incubated with 20 mg/L low-density lipoprotein receptor gene knockout(LDLr^-/-) mouse lipoprotein 20 mg/L ApoE gene knockout(ApoE^-/-) mouse lipoprotein or culture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blotting, respectively. RESULTS:The ApoE^-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addition, ApoE^-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A-I(ApoA-I) and high-density lipoprotein(HDL) compared with LDLr^-/- mouse lipoprotein treatment. ApoE^-/- mouse lipoprotein treatment inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr^-/- mouse lipoprotein treatment. CONCLUSION:Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.     

Retinoid X receptor agonist inhibits TGF-β1-induced collagen synthesis in cardiac fibroblasts by repressing Smad2 activation

AIM: To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms. METHODS: CFs were cultured using myocardial tissue with dry method. Hypoxic environment was established for CFs by continuous nitrogen supplement. Type I and type III collagens in supernatants were detected by ELISA. Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents. The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining. RESULTS: Under hypoxic condition, TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-dependent manner in the CFs. At the concentration of 5 μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01). RXR agonist 9-cis-retinoic acid (9-cis-RA; 10^-9~10^-6mol/L) decreased TGF-β1 (5 μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic condition. The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01). Smad2 inhibitor (20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF-β1 under hypoxic condition. Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly increased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05). CONCLUSION: Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition.     

Changes in plasma neurotension level of patients with coronary heart disease and its clinical significance

AIM: To investigate the plasma neurotensin(NT) concentration in patients with coronary heart disease: acute myocardial infarction(AMI),unstable angina(UA),stable angina pectoris(SAP), old myocardial infarction (OMI), and to study the relationship between the plasma NT level and the myocardial ischemia . METHODS:The plasma NT concentration of 30 patients with AMI,32 pat ients with UA,35 patients with SAP,31 pat ients with OMI and 32 normal controls were determined by radioimmunoassay(RIA). RESULTS: The plasma NT level in patients with AMI(24 h),in patients with UA when angina attacked is significantly higher than that of healthy controls. The plasma NT level in patients with SAP, in patients with OMI is not significantly different from that of healthy controls. The NT level of patients with UA when angina attacked is significantly higher than that after 2 weeks treatment (P＜0.01). The NT level of AMI patients rose from early period and reached peak value at 24 h .It began to decline at 48h,and restored to normal level at 72h. CONCLUSION: When acute myocardial ischemia occured in patients with coronary heart disease, the plasma NT level was elevated. This suggests NT may have participated in the pathophysiological course of myocardial ischemia.     

mRNA expression of insulin receptor and leptin receptor in liver of nonalcoholic fatty liver disease from diabetic rats

AIM: To observe the pathologic changes of liver in diabetic rats and to investigate the role of mRNA expression of insulin receptor and leptin receptor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). METHODS: Twenty male Sprague-Dawley rats were divided randomly into two groups: normal control group and diabetic group. After fed with high-fat diet for 4 weeks, diabetic rats were injected with streptozotocin at a dosage of 30 mg/kg intraperitoneally to induce NAFLD model of type 2 diabetes mellitus. Then the diabetic animals were fed with high-fat diet continuously for 12 weeks. At the end of the experiment, the rats were sacrificed, the concentrations of blood glucose, serum lipid, ALT and AST were measured biochemically. The levels of serum leptin and serum insulin were detected by enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA), respectively. The pathologic changes of liver were observed under light microscopy (LM) stained with HE, Sudan Ⅲ and Masson trichrome staining, respectively. The ultra-structural changes of liver were observed under transmission electron microscopy (TEM). Additionally, the mRNA expressions of PEPCK, G6Pase, insulin R and leptin R from rat livers were assayed by semi-quantitative RT-PCR. RESULTS: The levels of blood glucose, serum insulin, serum TG, ALT and AST increased significantly (P<0.01), serum TC elevated (P<0.05), and the levels of serum leptin decreased (P<0.01) in diabetic group compared to those in normal control group. Obvious liver fatty degeneration, piecemeal necrosis with accompanying inflammatory infiltration and fibrosis were found under LM. Hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of collagen in space of Disse were observed under TEM in diabetic group. The expression of insulin R and leptin R mRNA in liver from diabetic rats increased significantly (P<0.01) while the expression of PEPCK and G6Pase mRNA remained unchanged. CONCLUSION: Insulin resistance plays an important role in the pathogenesis of NAFLD. Low level of serum leptin, up-regulation of mRNA expression of insulin R and leptin R in liver caused by insulin resistance may be involved in this process.     

Expression of CXC chemokine receptor 7 in atherosclerotic ApoE-deficient mice and therapeutic impact by atorvastatin

AIM: To evaluate the expression level of CXC chemokine receptor 7 (CXCR7) in atherosclerotic apolipoprotein E-deficient (ApoE^-/-) mice induced by high-fat diet (HFD) and the effects of atorvastatin on it. METHODS: ApoE^-/- male mice (8-week-old) were used and were randomly divided into 3 groups following 1-week normal rodent diet: normal diet control (NDC) group, HFD group and HFD+statins (HFD+Sat) group. HE staining and oil red O staining were used to observe the atherosclerotic lesion burdens in the aortas. The expression of CXCR7 on the aortas was detected by Western blot and immunohistochemistry. The expression of Akt and endothelial nitric oxide synthase (eNOS) in the aorta was determined by Western blot.RESULTS: Few lesions were found in the aortas in NDC group. Apparent atherosclerotic plaque burdens were seen in HFD group and HFD+Sat group, while the atherosclerotic plaque burdens in HFD+Sat group were notably reduced compared with HFD group. The protein levels of CXCR7, eNOS and Akt in aorta in HFD group and HFD+Sat group were significantly decreased compared with NDC group, while those in HFD+Sat group were increased compared with HFD group. The protein level of p-eNOS in the aorta and the concentration of NO in the plasma in HFD group were decreased compared with NDC group and HFD+Sat group. CONCLUSION: In ApoE^-/- mice, HFD increases the lipid level and promotes the development of atherosclerosis by downregulating the expression of CXCR7, Akt and eNOS. Atorvastatin reverses the above effect of hypercholesterolemia on the expression of CXCR7, Akt and eNOS, thus playing the role in treating atherosclerosis.     

Study of platelet aggregations in patients with coronary heart disease and the effect of ticlopidine treatment

AIM: To investigate the platelet aggregations in patients with coronary heart disease(CHD) and the effect of ticlopidine treatment. METHODS: Platelet aggregations induced by adenosine diphosphate(ADP), epinephrine(EPI), collagen(Coll), arachidonic acid(ACA) in CHD group before and after ticlopidine treatment were measured by turbidity assay. RESULTS: Maximum ratios of platelet aggregations (max%) induced by ADP, EPI in CHD group (0.78±0.23, 0.86±0.25) were significantly higher than that of control group (0.65±0.19, 0.73±0.21, P＜0.05). After the treatment with ticlopidine, they were lowered obviously (0.68±0.18, P＜0.05;0.75±0.20, P＜0.05). There were no difference in max% induced by Coll and ACA between two groups and there were no significantly changes of max% induced by Coll and ACA by ticlopidine in CHD group. CONCLUSION: The platelet aggregations were increased in patients with coronary heart disease and could be inhibited by ticlopidine.     

Inhibition of Toll-like receptor 9 activation in microglia after oxygen-glucose deprivation and reoxygenation protects neurons from damage

AIM: To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation (OGDR), and its effects on neuronal apoptosis. METHODS: The BV-2 cell supernatants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h. The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant). The changes of the neuronal morphology were observed under an inverted phase- contrast microscope, and the neuronal apoptosis was detected by TUNEL. The protein expression of cleaved caspase-3 was detected by Western blotting. RESULTS: After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed. The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased. Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased. Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased. CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level. Inhibition of TLR9 expression reduces neuronal damage.     

Rheological properties and hemoglobin structure of red blood cells in cri-tically ill patients with coronary heart disease

AIM: To study the rheological properties of red blood cells (RBC) and the structure of hemoglobin in critically ill patients with coronary heart disease. METHODS: At pH 7.4, the rheological properties of RBC and the structure of hemoglobin isolated from critically ill patients with coronary heart disease and from healthy people were studied with both static and dynamic imaging techniques and ultraviolet-visible spectroscopy. RESULTS: The rheological properties of RBC from the critically ill patients with coronary heart disease were significantly different from those of healthy people. The bending modulus, which indicates the rigidity of RBC, was greater in the patients than that in healthy people. The absorption spectra of the hemoglobin of critically ill patients with coronary heart disease were almost the same as those of healthy people. CONCLUSION: The morphology and flexibility of RBC in critically ill patients with coronary heart disease are worse than those in healthy people, whereas the structure of hemoglobin in the patients is basically found no difference from that in healthy people.     

瓜菜类病原细菌耐药外排泵RND研究进展

近年来,瓜菜类作物病原细菌的耐药性日益增强,增加了瓜菜相关病害防治的难度。存在于瓜菜作物病原细菌中的外排泵耐药结节分化家族(RND)与细菌的耐药性及细菌/植物互作关系密切。笔者根据有关文献综述了RND外排泵的结构与功能、细菌/植物互作过程中外排泵的作用以及外排泵抑制剂(EPIS)的相关研究进展,为瓜类和蔬菜类作物病原细菌耐药性的研究提供参考。