Abstract:Aim To study whether plasma very low density lipoprotein (VLDL), low density Lipoprotein (LDL), high density lipoprotein (HDL) were oxidatively modified in endogenous hypertriglyceridemia and effects of VLDL, LDL and HDL on blood coagulation and fibrinolysis in vitro. Methods Plasma VLDL, LDL and HDL were isolated with density gradient ultracentrifugation method. Human plasma triglycerides (TG), total cholesterol (TC),high density lipoprotein cholesterol (HDLC) were measured by enzyme method. The oxidative modification of LDL, VLDL and HDL was identified by agarose gel relative electrophoretic mobility (REM), absorbance at 234 nm and fluorescence of thiobarbituric acid reaction substances (TBARS). Prothrombin time (PT),activated partial thrombplastin time (APTT), plasminogen activator inhibitor 1 (PAI 1) activity and tissue plasminogen activator (t PA) activity were measured in the reaction system consisted of freshly mixed normal plasma according to the direction of the kits. Results The hypertriglyceridemia (HTG) group were 1.6 and 0.4 times more plasma TG, TBARS level than the control group respectively (P<0.01). The plasma HDLC in HTG group was 32% lower than that of the control group (P<0.01). The rela tive electrophoretic mobility, absorbance at 234 nm and TBARS of VLDL, LDL and HDL in HTG group were significantly higher than that of the control group (P<0.01). The PT and APTT of VLDL, LDL and HDL in HTG group were significantly shorter than that of the control group (P<0.05). The t PA and VLDL in HTG group was higher and the PAI 1 in VLDL in HTG group was lower than that of the control group (P<0.01), but LDL and HDL were not influenced by the t PA and the PAI 1 in HTG group and the control group. The correlation analysis indicated that electrophoretic mobility of VLDL and HDL in HTG group was negatively correlated with PT (P<0.01). Conclusions Oxidative modification of plasma very low density lipoprotein, low density Lipoprotein, high density lipoprotein occurred in endogenous hypertriglyceridemia in vivo. LDL and HDL enhanced the activity of blood clotting system in vitro, but only VLDL affected the activity of fibrinolysis system.

Abstract:Aim To establish a new animal model which is more suitable for the study on atherosclerosis and to investigate its mechanisms and to study the immunological and inflammtional mechanisms in the pathogenesis of atherosclerosis. Methods 40 Wistar rats were randomly divided into 4 groups. Rats of the first group (n=10) were fed with high cholesterol diet (HCD). Rats of the second group (n=10) were fed with high cholesterol diet and injected with xenoma serum protein (HCD XSP). Rats of the third group (n=10) were fed with high cholesterol diet and injected with xenoma serum protein and adrenine (HCD XSP A), rats of the forth group (n=10) were fed with normal diet and just injected with adrenine (ND A). The changes of platelet function, lipid metabolism, leucocyte activation and adhesiveness, mediators of inflammation and blood vessel endothelial function were observed in four groups. Results In comparison with the ND A group, TC and LDLC of the HCD XSP A group rised significantly (p<0.01). As to the interleukins, the three groups fed with high cholesterol diet had high level of IL 6 and the HCD and HCD XSP groups had high level of IL 8, compared with the ND A group (p<0.01). But in all of the four groups, IL 2 did not have marked difference. The ND A group had the high leukocyte adhesion molecule express rate (CD11/CD18) than the other three groups (p<0.05). Conclutions The hyperlipidemia model induced by combined facters (HCD XSP A) is more suitable for the study on atherosclerosis and its mechanisms of traditional Chinese medicine treatment. And the immunological and inflammtional mechanisms play an important role in the pathogenesis of atherosclerosis.

Abstract:Aim To study the effect of anisodamine on lipopolysaccharide (LPS) induced expression of tissue factor (TF) in vascular endothelial cells (EC). Methods Human umbilical vein endothelial cells (hUVEC) were cultured by trypsin digestion method. TF activity was measured in the lysates of hUVEC by using a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. In order to evaluate a possible contribution of the nuclear factor κB (NF κB) pathway on anisodamine effects, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from hUVEC and NF κB binding oligonucleotides. Results Treatment of hUVEC with LPS resulted in a significant increase in TF activity. Anisodamine dose dependently inhibited LPS induced upregulation of TF. These effects were also confirmed on the level of specific TF mRNA expression by Northern blotting. Furthermore, anisodamine completely abolished LPS induced NF κB DNA binding activity in nuclear extracts from hUVEC treated with LPS together with anisodamine. Conclusions Anisodamine counteracts endothelial cell activation by inhibiting LPS induced TF expression in these cells. Its interference with the NF κB pathway might at least partly contribute to this effect. The ability of anisodamine to counteract the effect of LPS on endothelial cells might be one underlying mechanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.

Abstract:Aim To measurement the inhibitory effect of Momordicin on the activity of caspase 3 and apoptosis of myocardium in BALB/C mice with coxsackievirus B3 myocarditis. Method Momordicin was purificated from Momordica charantia L by our previous reported methods. Four group animals were used in this experiment, such as disease group (DG), normal group (NG), Momordicin group (MG), and Momordicin therapy group (MTG). Five animals were killed at the 3rd,the 7th,and the 14th days in each group, and all were killed at the 21st day. According to the method of Calbiochem Company, the activity of caspase 3 from the heart of BALB/C mice was measured, and apoptosis was measured by terminal transferase mediated DNA nick end labeling assay (TUNEL, the method provided by Oncogene Company). Results ①The activity of caspase 3 were measurable at the 7th day in the DG (0.63±0.21 pmol/min, n=5). The activity of caspase 3 at the 14th day (10.9±1.5 pmol/min, n=5) and the 21st day (12.6±1.3 pmol/min, n=5) are higer than the 7th day, and the activity of the 21st was higher than 14th day (p<0.05). ②Only a animal has the activity of caspase 3 at the 21st day (0.41 pmol/min) in the HTG, no caspase 3 activity were detected in the animals of MG and NG. ③Only a few apoptotic cells were found at the 7th day in the DG, and more apoptotic cells were found among cardiac muscles at 14th and 21th days. Single apoptotic cardiomyocytes were also found at the outside of the pathological change areas, and no apoptotic cells were found in the animals of NG, MG, and MTG. Conclusions Distinct apoptosis were found in CVB3 viral myocarditis; the activitys of caspase 3 and apoptosis were found at the 7th day, and higher after the 7th day, single apoptotic cardiomyocytes were also found. The high dose Momordicin has distinct inhibitory effect on the activity of caspase 3 and apoptosis.

Abstract:Aim To investigate the effects of the scavenger receptor A_Ⅰ( SR A_Ⅰ) on expression of matrix metalloproteinase 2 (MMP 2) and matrix metalloproteinase 9 (MMP 9) and its tissue inhibitor of metalloproteinase (TIMP) in macrophage drived foam cells. Methods THP 1 cells were incubated with PMA, then added oxidized low density lipoprotein (ox LDL) to induce macrophage cell foaming. Foamed macrophage cells were verified by oil red staining and high performance liquid chromatography (HPLC). Scavenger receptor A_Ⅰ mRNA was detected by reverse transcriptase polymerase chain reaction (RT PCR). Antisense and antibody were used to block SR A_Ⅰin foamed cell. MMP 2, MMP 9, TIMP 1 and TIMP 2 were detected by Western blot. MMP 2 and MMP 9 activities in media were estimated using zymography. Results Blocking SR A_Ⅰinhibits activities and expression of MMP 2 and MMP 9, increases the expression of TIMP 1 and TIMP 2. Conclusions Blocking the activities of SR A_Ⅰmay contribute to maintain the balance between MMP and TIMP and inhibit macrophage mediated matrix breakdown in the atherosclerotic plaques.

Abstract:Aim To explore the effect of oxidized low density lipoprotein(ox LDL) on cholesterol accumulation in mouse peritoneal macrophages and its relationship with activity of lysosomal cathepsin. Methods Low density lipoprotein (LDL), acetylated LDL (ac LDL) and oxidized LDL (ox LDL) were prepared and treated the macrophages in the same way. Lysosome and cytoplasm were acquired from the treated macrophages and the contents of total protein,cholesterol and the activities of lysosomal cathepsin L and D were determined. Results Compared to control, both treatment of ac LDL and ox LDL significantly increased the cholesterol level in macrophages (22.2±0.4 μg and 22.55±0.15 μg to control 7.0±0.4 μg, p<0.01), however, ox LDL and ac LDL resulted in cholesterol accumulation in different manner. The ac LDL caused cholesterol ester (CE) accumulation predominating in cytoplasm (18.9±0.4 μg and 3.33±0.20 μg respective in cytoplasm and lysosome, 0.73±0.05 μg and 18.1±0.4 μg respective in FC and CE form), while ox LDL leaded free cholesterol (FC ) accumulation preferring in lysosome of macrophages (3.65±0.14 μg and 18.90±0.15 μg respective in cytoplasm and lysosome, 14.13±0.14 μg and 4.77±0.33 μg respective in FC and CE form) and simultaneously we found that ox LDL markedly inhibited the activities of cathepsin L and D (in the unit of a.u/mg pro, 11.1±2.3 and 2.1±0.5 to control 102.3±8.2 and 27.33 ±1.6,p<0.01); and gave rise to the content of total protein in lysosome. Conclusion Oxidized LDL cause the cholesterol accumulation in lysosome mainlly in free manner and this effect may be related to its effect of inhibiting the activities of cathepsin L and D.

Abstract:Aim To evaluate the therapeutic effects of simvastatin,probucal,captopril and chinese medicine on endothelium function of atherosclerosis(As). Methods The rabbits of As were given simvastatin, probucal, captopril and chinese medicine respectively for 12 weeks. The blood were drawn for measuring the lipids,malondialdehyde, superoxide dismutase, nitric oxide and endothelin 1 (ET 1). The endothelium dependent vascular relaxation (EDVR) of abdominal aorta was examinaed by injecting acetylcholine (Ach). The mRNA expression of inducible nitric oxide synthease (iNOS), ET 1, angiotensin converting enzyme (ACE) of As were examined by reverse transcription polymerase chain reaction. Results The aortic contraction caused by Ach decreased in probucal, simvastatin, chinese medicine and captopril groups than regression control group. Both total cholesterol and intima medial thickness showed negative correlations with EDVR(r=-0.57,0.47 respectively). In normal group, mRNA of iNOS, ET 1 and ACE were expressed in low levels. In comparison with regression control group, mRNA expression of iNOS,ET 1 and ACE in 4 drug therapy groups decreased, iNOS in probucal and captopril groups and ACE in probucal, captopril and chinese medcine groups reduced. Conclusion The 4 drugs can improve the EDVR disfunction in atherosclerosis and probucol has the best effect. EDVR improvement may be associated with the mechanisms of reducing expression of iNOS, ET 1 and ACE genes, and reduction of lipid peroxide.

Abstract:Aim To evaluate the effect and feasibility of domestic afterloading system in the prevention of restenosis after vascular angioplasty. Methods Atherosclerotic models in domestic microswine were established with the standard method of vascular injury plus hypercholesterol food. Angioplasty with oversized balloon was undertaken in bilateral internal and external iliac arteries in 16 weeks. The target vessels were randomized in to irradiation group and control group. 192 Ir afterloading system were employed for the endovascular irradiation therapy in irradiation group, and the radiation dose was 20～25 Gy. The target vessels were harvested for the study of vascular remodeling at the end of the 12th week and the 24th week respectively. Results The areas of intima and media were smaller, but the residual cavity areas were larger significantly in irradiation group than in control group (p<0.01, 0.05, 0.01, respectively). However, there was no significant difference between the two areas beneath external elastic lamina (p>0.05). Conclusion Endovascular irradiation with 192 Ir afterloading system might reduce restenosis by effecting the vascular remodeling after angioplasty.

Abstract:Aim To observe the effect of oxidized low density lipoprotein (ox LDL) on the expression and activity of matrix metalloproteinases(MMPs). Methods Human monocyte derived macrophages were cultured in vitro and the gene and protein expressions of MMPs (MMP 2 and MMP 9) were detected by reverse transcription polymerase chain reaction and Western blot respectively. MMPs'activity was detected by Zymography. Results The results revealed that 10,20 and 40 g/L of ox LDL could stimulate the gene expression of MMP 2 and MMP 9 in comparison with the same concentrations of LDL. The expressions of MMP 2 protein and MMP 9 protein were higher in ox LDL groups than in LDL groups. The activity of MMP 2 and MMP 9 was also significantly higher in different concentrations of ox LDL groups than the same concentrations of LDL groups. The activity of MMP 2 and MMP 9 was in a dose dependent manner in ox LDL group. Conclusions The results indicate that ox LDL can obviously increase the expressions and activities of MMP 2 and MMP 9 in human monocyte derived macrophages. So ox LDL could enhance the breakdown of matrix in lesions of atherosclerosis which could probably induce vulnerable plaque. Rupture of the vulnerable plaque is the major cause of acute coronary events.

Abstract:Aim To investigate the effect of losartan on neointimal proliferation and expression of nuclear factor κB(NF κB). Methods Japanese White rabbits underwent abdominal aorta balloon de endothelialization and then were treated with a 1.5% cholesterol diet for 8 weeks. Then balloon angioplasty was performed on the injured arteries. Immediately after balloon angioplasty, the rabbits of the losartan group was orally administered with losartan [10 mg/(kg·d)], while the rabbits of the control group were given normal saline. Animals were killed four weeks after angioplasty, and excised artery segments were prepared for histomorphological observation and α smooth muscle actin, macrophage, NF κB and intracellular adhesive molecule 1 (ICAM 1) were investigated by immunohistochemistry analysis. Results Losartan treatment significantly inhibited neointimal proliferation. Compared with the control group, the intimal thickness (IT), intimal area (IA), the ratio of IT/MT and IA/MA of the losartan group were significantly reduced (p<0.01). Also, macrophages and smooth muscle cells (SMC) in the neointima were significantly diminished (p<0.01 and p<0.05) and the expression of NF κB and ICAM 1 were respectively decreased in the losartan group (p<0.01 and p<0.05). Conclusions These findings suggest that losartan could significantly relieve neointimal proliferation after balloon angioplasty, possibly through inhibiting angiotensinⅡ and NF κB, thus inhibits inflammatory reaction, migration and proliferation of SMC as well as formation of neointima which contributes to restenosis after balloon angioplasty.

Abstract:Aim To investigate the effect of aldosterone on expression of angiotensin Ⅱ type 1 (AT1) receptor in rat cardiac fibroblasts (CFs) and test the effect of aldosterone on AngⅡ induced collagen synthesis. Methods Cardiac fibroblasts culture were established from neonatal rats. Collagen synthesis was measured by 3 H proline incorporation after incubation with AngⅡ, aldosterone and AngⅡ plus aldosterone. The density of AT 1 recetpor was determined by 125 Ⅰ Ang Ⅱ saturation binding, and AT1 receptor mRNA levels were analyzed by quantitative reverse transcriptase polymerase chain reaction(RT PCR). Results AngⅡ increased collagen synthesis in cultured CFs; while aldosterone did not affect production of collagen. However, aldosterone could significantly potentiate the incremental effect of AngⅡ on collagen synthesis in CFs. The density of AT1 receptor inceased 2 fold and was accompanied by a 1.5 fold increase in the corresponding mRNA after treatment of aldosterone(10 -7 mol/L). Conclusion Aldosterone does not exert a direct effect on collagen synthesis in cultured rat CFs, but could influence the collagen output of CFs through the upregulated AT1 receptor.

Abstract:Aim To evaluate the effect of simvastatin on interleukin 6 (IL 6) production in human monocytes stimulated by C reactive protein (CRP) and lipopolysaccharide (LPS), assess the influence of simvastation on monocytes inflammatory reaction. Methods Monocytes were isolated by ficoll density gradient from blood of healthy volunteers. To measure interleukin 6 production(by ELISA) stimulated by 20 mg/L CRP and 10 μg/L LPS at 2 h, 4 h, 8 h, 16 h, 24 h or 1 mg/L, 5 mg/L, 10 mg/L, 20 mg/L CRP and 1 μg/L, 2.5 μg/L, 5 μg/L, 10 μg/L LPS at 24 h, the peaks to be compared with which inhibited by simvastatin(10 -8 mol/L～10 -6 mol/L). Results Interleukin 6 could be produced after being stimulated with 20 mg/L CRP at 4 h and with 10 μg/L LPS at 2 h. The effects of CRP and LPS were dose dependent and time dependent, reaching peaks at 24 h. The highest levels of monocytes IL 6 were 904±77 ng/L, 1 654±765 ng/L in CRP and LPS groups, respectively. Simvastatin (10 -8 mol/L～10 -6 mol/L) inhibited IL 6 production induced by 20 mg/L CRP in a dose dependent manner. Only 10 -6 mol/L simvastatin could inhibit IL 6 production induced by 10 μg/L LPS. Conclusion C reactive protein and LPS induce the production of IL 6 in human monocytes; simvastatin can inhibit the production of IL 6, it may be used in clinical prevention and treatment of coronary heart disease.

Abstract:Aim To determine whether left ventricular remodeling with or without onset heart failure after myocardial infarction is associated with the regional changes in the gene expression of fatty acid metabolism. Methods Myocardial infarction(MI) was induced in rats by ligation of the left anterior descending coronary artery. Hemodyanmics, ventricular remodeling parameters were investigated in the experimental and sham operated animals(SH) at 2, 4, 8 week after operation. In infarcted hearts, the peri infarction region(4 mm zone surrounding the region) were separated for gene expression analysis of the rate limiting enzyme[muscle carnitine palmitoyltransferase I (M CPT I)], key enzyme[medium chain Acyl CoA dehydrogenase(MCAD)] of fatty acid oxidation and nuclear transcription factor [peroxisome proliferator activated receptors α(PPAR α)]. Results At 2 weeks after LAD ligation, when right ventricular hypertrophy was present without signs of heart failure, mRNA expression of M CPT I, MCAD and PPAR α was reduced in the peri infarction regions[27%,35%and 20% respectively in MI 2w vs SH; ([WTBX]p<0.05)]. Until 8 weeks, left ventricle displayed a little hypertrophy with heart failure, there was significant downregulation in mRNA expression of M CPT I, MCAD and PPAR α in the peri infarction regions . Until 8 weeks, left ventricle displayed a little hypertrophy with heart failure, there was significant downregulation in mRNA expression of M CPT I, MCAD and PPAR α in the peri infarction regions [reduced 52%,60% and 44% respectively in MI 8w /SH 8w vs MI 2w /SH 2w ;([WTBX]p<0.05)]. . Conclusion In rats with MI, progression from compensated remodeling to heart failure is associated with gene expression downregulation of fatty acid oxidation in the peri infarction region. Downregulation of PPAR α mRNA displays that PPAR α may be a very important factor that regulates cardiac fatty acid oxidation in rats subsequent to myocardial infarction.

Abstract:Aim To evaluate the effect of LDL and ox LDL on the activity and mRNA expression of hepatic lipase (HL) in HepG2 cells. Methods Gradient concentrations (1.7～1 758 mg/L) of LDL or ox LDL was added into the medium and coincubated with HepG2 cell. The cytostasis rate of HepG2 cell was estimated by MTT and the activity and mRNA expression of HL in these cells were measured with cytochemistry and RT PCR respectively. Results In the 27.47 mg/L and 54.95 mg/L LDL groups, the mRNA expression of HL was up regulating 2.8 times and 2.3 times respectively to the cell control. In the 109.8 mg/L LDL, the mRNA expression of HL decreased 20% to the cell control. The HL activity in HepG2 cells incubated with LDL was negative related with the LDL concentration (r=-0.95614, p<0.05). The cytostasis rate in LDL increased by dose dependent model (r=0.91199, p<0.05). In the ox LDL group, the mRNA expression of HL was inhibited at all concentrations. In 27.47 mg/L and 54.95 mg/L ox LDL, the activity of HL was 3～4 times as much as that of LDL groups. Moreover, at the same concentration of 54.95 mg/L, the cytostasis rate in the ox LDL group was one time higher than in the LDL group. Conclusions At the appropriate range of concentration, LDL can induce the up regulation of mRNA expression of HL. High concentration of LDL and all concentration of ox LDL can inhibit not only mRNA expression but the activity of HL in HepG2 cells.

Abstract:Aim To investigate the relationships between essential hypertensives and the polymorphism of angiotensin II type 1 receptor (AT1R) gene in youth. Methods 104 hypertensive and 154 normotensive subjects were studied. All subjects were divided into four groups according the age: young hypertensives, aged hypertensives, young and aged normotensives. The A→C variant at position 1166 (A1166C) of the AT1R gene was identified by polymerase chain reaction (PCR) and PCR/restriction fragment length polymorphism (PCR/RFLP) analysis. The frequencies of AC+CC genotypes and 1166C allele of AT1R gene between hypertensives and normotensives were analyzed by Chi Square test. Results The frequencies of AC+CC genotypes and 1166C allele of AT1R gene in hypertensives were higher than those in control (AC+CC genotypes 0.202∶0.097, p<0.05; C allele 0.115∶0.052, p<0.01), the OR(odds ratio)is 2.34(95%CI: 1.15～4.67). More statistically significant of C1166 allele frequency and the AC+CC genotype distribution were obtained in youth between the case and the control when age was hierarchically analyzed (AC+CC genotypes 0.217∶0.088,p<0.01; C allele 0.183∶0.044,p<0.01), the OR reach to 6.82(95%CI: 2.02～23.08). Further more, in the case group, the frequencies of AC+CC genotypes and 1166C allele of AT1R gene in youth were higher than those in middle aged and the aged. Conclusions 1166 AC+CC genotypes and 1166C allele of AT1R gene were related to essential hypertension and could increase the fatalness of suffering from hypertension in youth. It plays an important role in patients who suffered from premature essential hypertension.

Abstract:Aim To study the changes in endothelium dependent vasodilation function,serum nitric oxide (NO) and superoxide dismutase (SOD) and their inter relationships in patients with essential hypertension (EH). Methods High resolution B mode ultrasonography was used to measure the endothelial dependent vasodilation in 28 patients with EH and 28 matched healthy subjects. Serum levels of NO and SOD were also measured. Results The increased percentage of the brachial artery diameter after reactive hyperemia was lower in patients with EH than that of normal controls (6.33%±2.59% vs 13.14%±4.24% p<0.001), whereas nitroglycerin induced changes were similar (p>0.05). Serum levels of NO,SOD in patients with EH was significantly lower compared with those in controls (53.55±23.5 μmol/L vs 83.26±23.25 μmol/L,86.57±26.66 kNU/L vs 117.1±33.64 kNU/L,p<0.001). Correlation analysis shows that endothelium dependent vasodilation function was negatively correlated with ages,systolic blood pressure,dilated blood pressure,the coefficients were -0.366 (p<0.01),-0.65 (p<0.001),-0.672 (p<0.001),and was positively correlated with serum NO and SOD,the coefficients were 0.838 (p<0.001),0.683 (p<0.001). The analysis of multiple stepwise regression shows that the endothelium dependent vasodilation function was negatively correlated with dilated blood pressure and positively correlated with serum NO and SOD. Conclusion Our findings support that endothelium vasodilation is impaired and was correlated with reduction of NO and increase of active oxygen species.