Abstract:Aim To detect the methylation and expression of CpG island in the gene promoter region of live CuZnSOD during the occurrence of atherosclerosis(As) on apolipoprotein E-deficient mouse,explore the effects of betaine on atherosclerosis and its possible mechanisms.Methods Normal C.57BL/6J mouse were taken as control group,apolipoprotein E-deficient mouse were randomly divided into model group,1%,2%and 3%betaine group.Oil red O staining method was used to detect the lipid plaque area in mouse aortic sinus,methylation-specific polymerase chain reaction (MSP) was applied to examine the gene methylation of liver CuZnSOD,quantitative fluorescence reverse transcription-polymerase chain reaction(real-time RT-PCR) was taken to detect the expression of mRNA in CuZnSOD gene and by immunohistochemical method its protein expression was detected.Results In 14th week,the lipid plaque areas of aortic sinus in 2%and 4%betaine mouse group were less than the model group,the difference was significant(P<0.05);The CpG island methylation status of CuZnSOD gene in each group had no significant difference(P>0.05);in all time the expression of mRNA in the normal groups were higher than that in the model groups;the 7th week,the expression of mRNA in 1%betaine group was higher than that in 2%and 4%betaine group;the 14th week,the expression of mRNA in 1% betaine group was higher than that in model group and 2%betaine group.In all time the expression of CuZnSOD protein in the normal group was higher than that in the other four groups(P<0.05),but the expression in the other four groups had no signifcant difference.Conclusions CuZnSOD gene may not be involved in the abnormal DNA methylation changes in the process of atherosclerosis,supplement of betaine can increase the expression of CuZnSOD gene mRNA in liver,thus improve the lipid aggradation of apolipoprotein E-deficient mouse and reduce the plaque area of aortic.

Abstract:To analyze the basic character of a novel Homosapiens synapse associated protein gene(FRG4) with bioinformatics.FRG4 full-length sequence was obtained by hot-start PCR from human fetal liver library and its genomic constitution,the multi-sequences homology,the transmembrane domains,hydrophilicity and hydrophobicity,as well as its functional and structural domains were analyzed and predicted by bioinformatics.We have successfully attained the fulllength cDNA sequence of FRG4,bioinformatic analysis shows FRG4 gene has 99%homology with Homosapiens synapse associated protein 1(SYAP1),consists of 9 exons and 8 introns and located in Xp22.2.None transmembrane domains but a BSD domain was discovered,and its encoding protein is probably a water-solubility protein.

Abstract:Aim To evaluate the effect of water extract propolis(WEP) on human umbilical artery smooth muscle cell proliferation(HUASMC) induced by angiotensinⅡ(AngⅡ) in vitro and explore the underlying mechanism. Methods HUASMC were cultured by the explant method and incubated with 50 mg/L,100 mg/L and 200 mg/L WEP. HUASMC number was assessed by cell counting;Flow cytometry was used to test cell cycle;Proliferating cell nuclear antigen (PCNA) of HUASMC was detected by immunocytochemistry staining;Superoxide dismutase(SOD) and maleic dialdehyde (MDA) in HUASMC culture medium superior was measured with spectrophotometer;Apoptosis was evaluated by terminal -deoxynucleotidyl transferase mediated nick end labeling(TUNEL).Results Compared to model group,the numbers,PCNA expression,MDA,Apoptosis Index of 100 mg/L and 200 mg/L WEP were lower(P<0.01),while the Percentage of the Cells in G_0/G_1 phase and SOD was higher(P<0.01).Conclusion Certain concentration of WEP can prevent HUASMC proliferation effect mediated by AngⅡ;the mechanism about WEP effect may be associated with arresting G_1 to S progression,decreasing PCNA expression and improving the antioxidation of HUSMC.

Abstract:Aim To investigate the effect of adrenaline on the expression of ATP binding cassette transporter A1 (ABCA1) mRNA and scavenger receptor AI(SR-AI) mRNA in THP-1 macrophages and to identify its possible mechanism mediated byβ-adrenoceptor.Methods THP-1 derived macrophages were preincubated for an hour with aβ1-adrenoceptor antagonist metoprolol(10 nmol/L to 100μmol/L) and aβ2-adrenoceptor antagonist butoxamine(2.5 nmol/L to 25μmol/L),respectively,before treatment with 1μmol/L adrenaline for 24 h.Then ABCA1 and SR-AI mRNA expression were determined by RT-PCR.Results RT-PCR analysis showed that compared with control(without adrenaline),1μmol/L adrenaline significantly decreased ABCA1 mRNA expression and significantly increased SR-AI mRNA expression (P<0.05).These stimulatory effects of adrenaline on ABCA1 mRNA and SR-AI mRNA expression in THP-1 macrophages were reversed by theβ1-adrenoceptor antagonist metoprolol or theβ2-adrenoceptor antagonist butoxamine in a concentration -dependent manner.Furthermore,the effect of theβ2-adrenoceptor antagonist butoxamine reached a platform at the concentration of 2.5μmol/L.Conclusion Adrenaline up-regulates SR-AI mRNA expression and down-regulates ABCA1 mRNA expression,probably being mediated viaβ1-adrenoceptor orβ2-adrenoceptor activation.

Abstract:Aim To examine the expression of NARC-1 protein in atherosclerotic lesion of New Zealand rabbits. Methods Male purebred New Zealand rabbits with adaptation feeding for 1 week,were randomly divided into control group(n = 8) and high-cholesterol group(n = 8).The control group was given a normal control diet and the high-fat group was fed a high-cholesterol diet(2%cholesterol).All rabbits were euthanized at the end of the 8-week experiment. Plasma triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDLC) and low density lipoprotein cholesterol(LDLC) were determined by commercially enzymatic methods.Atherosclerosis index(AI) and the HDLC/LDLC ratio were calculated.SudanⅣstaining was used to examine the aortic atherosclerotic lesions.The pathological changes of rabbit aortic preparations was quantified by computer-assisted image analysis of oil red O and HE staining using Image-Pro Plus image analysis software.NARC-1 protein distribution and expression was analyzed by immunohistochemistry and Western blotting.Results Plasma TC,HDLC and LDLC concentrations,and AI were markedly increased, but the HDLC/LDLC ratio was markedly decreased in high-cholesterol group.High-cholesterol diet induced significant aortic atherosclerotic lesions.Histopathology analysis showed that aortic intima was obviously thickened and the intima/media thickness ratio was significantly increased in high-cholesterol group rabbits.Oil red O staining indicated that the number of foam cells in artery endothelium were markedly increased in the high-cholesterol group compared with the control group.HE staining revealed the aortic intima in control rabbits was intact and thin,compared to thickened aortic intima and remarkable atherosclerotic plaques in high-fat group rabbits.Immunohistochemical detection showed NARC-1 protein was found in the intima plaque and located in cytoplasma and plasma membrane of foam cells in high-fat group. Western blotting analysis also indicated NARC-1 protein expression was significantly elevated in the aortic atherosclerotic plaque of high-fat group rabbits.Conclusion The high-cholesterol diet induced aortic atherosclerotic lesion in New Zealand rabbits.NARC-1 protein was remarkably expressed in aortic atherosclerotic plaque and located in the cytoplasma and plasma membrane of the foam cells in high-fat group rabbits.The results indicate that NABC-1/PCSK9 is one of the important factors involved in atherosclerosis development.

Abstract:Aim To observe the effect of amlodepine on pregnancy-associated plasma protein-A in atherosclerosis. Methods 20 male New Zealand rabbits were fed on a diet of 1%cholesterol for 12 weeks after they underwent ballon-induced abdominal arotic wall injury.At the end of the 12th week,they were divided into 2 groups at random. One group was given Amlodipine(5 mg/d) for 12 weeks,the other was control group.At the beginning of the experiment, the end of 12th week and 24th week,blood samples were collected to measure the concentrations of pregnancy-associated plasma protein-A and high sensitive C-reaction protein;at the same time,abdominal aorta were examined by high frequency ultrasound.At the end of 24th week,abdominal aorta were processed and examined by pathological stain and immunohistochemical stain for pregnancy-associated plasma protein-A.Results Pathological results and electron microscope showed that compared with control group,the plaque area and Lipid deposition of amlodipine group reduced, smooth muscle cells increased and collagen minished.Ultrasound measurements also showed that intima-media thickness and end systolic diameter were significantly decreased(P<0.01) and corrected intima-media echo intensity was increased (P<0.01) in amlodipine group.Amlodipine also decreased pregnancy-associated plasma protein-A and high sensitive Creaction protein in blood(P<0.01) and pregnancy-associated plasma protein-A expression in atherosclerotic plaque with immunohistochemical stain.Pregnancy-associated plasma protein-A had a close relationship with high sensitive C-reaction protein and intima-media thickness(R~2 = 0.424 and R~2 = 0.339).Conclusion Pregnancy-associated plasma protein -A is associated with severity of atherosclerosis.Amlodipine can regress atherosclerosis by inhibiting pregnancy-associated plasma protein-A.

Abstract:Aim To investigate the effect of 7-difluoromethyl-genistein(FMGEN) on oxidative stress-induced cell adhesion between vascular endothelial cells and mononuclear cells and the underlying mechanism.Methods Fluorescent light spectrophotometer was used to detect the cell adhesion between vascular endothelial cells and mononuclear cells.The concentrations of E-selectin and intercellular adhesion molecule(ICAM-1) in the cell culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA).The activation of P38 mitogen-activated protein kinase (P38-MAPK) was analyzed by Western blotting.Results Exposure of vascular endothelial cells to H_2O_2 for 24 h increased the adhesion between vascular endothelial cells and mononuclear cells and the release of E-selectin and ICAM-1 in vascular endothelial cells;however,these effects of H_2O_2 were inhibited by FMGEN in a concentration-dependent manner. Treatment of vascular endothelial cells with H_2O_2 for 24 h resulted in the significant activation of P38-MAPK and the activation of P38 induced by H_2O_2 was inhibited by FMGEN.SB203580,a specific inhibitor of P38-MAPK blocked the adhesion between vascular endothelial cells and mononuclear cells and the release of E-selectin and ICAM-1 in vascular endothelial cells induced by H_2O_2.Conclusion FMGEN antagonizes oxidative stress-induced cell adhesion between vascular endothelial cells and mononuclear cells,which is associated with inhibition of the release of E-selectin and ICAM-1 via down-regulating the activation of P38-MAPK.

Abstract:Aim To investigate whether the expression of groupⅣcytosolic phospholipase A2(cytosolic phospholipase A2,cPLA2),groupⅤsecretory phospholipase A2(sPLA2-Ⅴ) and groupⅩsecretory phospholipase A2 (sPLA2-Ⅹ) could be regulated by oxidized low density lipoprotein(ox-LDL) of different oxidation levels in the mouse macrophage cell line RAW264.7 cells,and to explore the inflammatory mechanisms of the interactions between ox-LDL and macrophages in the process of promoting atherosclerosis.Methods 3 types of ox-LDL(minimally modified low density lipoprotein or MM-LDL,lightly ox-LDL or ox_(4h)-LDL,heavily ox-LDL or ox_(48h)-LDL) were prepared by different oxidation ways.3 types of ox-LDL were used to incubate with RAW264.7 cells for 4 h and 8 h,then the cell lysate and cell culture supernatant were collected.The mRNA expression and protein expression of cPLA2,sPLA2-Ⅴand sPLA2-Ⅹwere determined respectively by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting analysis. Results The mRNA expression and protein expression of sPLA2-Ⅴand sPLA2-Ⅹwere undetectable in untreated RAW264.7 cell.Compared with the PBS control group and the LDL group,the mRNA expression of cPLA2 was not affected by all types of ox-LDL(P>0.05).But the mRNA expression and protein expression of sPLA2-Ⅴand sPLA2-Ⅹwere significantly enhanced by the stimulation of MM-LDL and ox_(4h)-LDL in RAW264.7 cell(P<0.05),while ox_(48h)-LDL had a slight ability to induce the expression of sPLA2-Ⅴand sPLA2-Ⅹ(P<0.05).However,PBS control group and native LDL failed to induce the expression of sPLA2-Ⅴand sPLA2-Ⅹ.Conclusion The exposure of ox-LDL to RAW264.7 cells did not affect the mRNA expression of cPLA2,but could significantly induce RAW264.7 cells to express sPLA2-Ⅴand sPLA2-Ⅹ.Two types of mildly oxidized LDL(MM-LDL and ox_(4h)-LDL)-induced expression of sPLA2-Ⅴand sPLA2-Ⅹwas stronger than that of heavily oxidized LDL(ox_(48h)-LDL),indicating that mildly oxidized LDL was capable of inducing a strong inflammatory response on macrophages.

Abstract:Aim To explore the effect of endothelial progenitor cells(EPC) on the phenotype transformation of cultured rat vascular smooth muscle cells(VSMC) stimulated by angiotensinⅡ.Methods Mononuclear cells were isolated from fresh cord blood by 3%Gelatin and density gradient centrifugation.Isolated cells were cultured in EGM-2 medium supplemented with 20%FBS,VEGF,bFGF and other growth factors.Biological features of EPC were observed at different time points,and EPC was identified by morphology,fluorescence double-staining and flow cytometry.Indirect immunofluorescence was performed to analyze expression ofα-SM-actin,calponin of VSMC special antigen.RT-PCR and Western blotting were performed to analyze the effect of endothelial progenitor cell conditional medium(E-EPC-CM,LEPC -CM) and human umbilicus vein endothelial cell conditional medium(HUVEC-CM) on AngⅡ-induced expression ofα-SM-actin and osteopontin in VSMC.VSMC was divided into control group,AngⅡgroup,AngⅡ+EGM-2 group,AngⅡ+E-EPC-CM group,AngⅡ+L-EPC-CM group and AngⅡ+HUVEC-CM group.Results After stimulation with angiotensinⅡfor 48 h,the expression of theα-SM-actin mRNA and protein significantly decreased and the osteopontin mRNA and protein markerly increased compared with control group,suggesting that VSMC was changed from contractile to synthesize type by angiotensinⅡ.Treatments with E-EPC-CM,L-EPC-CM,HUVEC-CM,especially with E-FPC-CM, could inhibit significantly the down-regulation ofα-SM-actin expression and the up-regulation of osteopontin expression by AngⅡ.Conclusion EPC could inhibit the phenotype transformation of VSMC from contractile to synthesize type induced by angiotensinⅡ.

Abstract:Aim To investigate the effects of Angiotensin(1-7)(Ang(1-7)) on the expression of lectin-like oxidized low density lipoprotein receptor-1(LOX-1) in human umbilical vein endothelial cells(HUVEC) induced by angiotensinⅡ(AngⅡ).Methods HUVEC were isolated and cultured.Cultured HUVEC were incubated for 24 h with Ang-(1-7),AngⅡ,Ang(1-7)+A-779,Ang(1-7)+AngⅡ,A-779+AngⅡ+Ang(1-7),respectively.And cultured HUVEC without incubating stimulator were chosen as controls.The expression of the mRNA and protein of LOX- 1 was measured by RT-PCR and flow cytometry methods respectively.The expression of the phosphorylation of p38 mitogen activated protein kinase(MAPK) was determined by Western blotting.Results Compared with the AngⅡgroup, Ang(1-7) dose-dependently inhibited the expression of LOX-1 in HUVEC(P<0.05).Compared with control group, the expression of the phosphorylation of p38 MAPK increased significantly in AngⅡgroup(P<0.05),while it was a little in Ang(1-7) group and A-779 group(P>0.05).The expression of the phosphorylation of p38 MAPK reduced in AngⅡ+Ang(1-7)group compared with AngⅡgroup(P<0.01).The expression of the phosphorylation of p38 MAPK had no significant change in AngⅡ+Ang(1-7)+A-779 group(P>0.05).Conclusion Ang(1-7) concentration dependently attenuate the expression of LOX-1 induced by AngⅡin HUVEC through its specfic receptor MAS,Ang(1-7) could inhibit the expression of the phosphorylation of p38 MAPK induced by AngⅡin HUVEC,and then inhibit the AngⅡ-induced LOX-1 espression.

Abstract:Aim To determine the effective concentration of remifentanil in 50%essential hypertensive patients (EC_(50)) for blocking the sympathetic activation induced by desflurane.Methods Thirty essential hypertensive patients scheduled for elective surgery were enrolled in the study.The patients were inspired with desflurane 0.5 MAC after endotracheal intubation.Twenty minutes later,the first dose of remifentanil 4μg/L which was determined by up and down sequential experiment was given by target plasma concentration(TCI).Both the blood perssure(BP) and heart rate (HR) were recorded and averaged every 30 seconds of 3 minutes before or after fast inducting desflurane 1.5 MAC.If both the rate of average BP and average HR were increase 15%the next patient received remifentanil of a lower concentration; if both the rate of average BP and average HR were descrease 15%the concentration of remifentanil was increased in the next patients.Result Among the 30 essential hypertensive patients,five were excluded from the study because of uncertain efficacy.The results showed that the EC_(50) of remifentanil blocking the sympathetic activation induced by desflurane was 2.09μg/L(95%CI was 2.08μg/L～2.10μg/L).Conclusions The EC_(50) of remifentanil given by TCI which blocked the sympathetic activation induced by desflurane in essential hypertensive was 2.09μg/L determined by sequential experiment method.

Abstract:Aim To measure the plasma soluble OX40 ligand(sOX40L) levels and carotid intima-media thickness (IMT) in patients with coronary heart disease and age-,sex-matched controls,trying to investigate the relationship between sOX40L and coronary heart disease.Methods Enzyme-linked immunosorbent assay was used to measure plasma sOX40L in 35 patients with stable angina pectoris,30 individuals with high risk factors of coronary heart disease, and 20 age-,sex-matched healthy controls.Carotid IMT was measured with carotid ultrasonography.Results The levels of sOX40L(24.95±15.60 ng/L) was significantly higher in patients with stable angina pectoris compared with control group(16.44±11.31 ng/L;P<0.05).Patients with stable angina pectoris and risk factors had higher values of carotid IMT than controls.Multiple regression analysis indicated that sOX40L level was an independent contributor to carotid IMT(P = 0.019) after controlling age,lipid levels,and blood pressure.Conclusion sOX40L level is an independent contributor to carotid IMT,and suggests the potential diagnostic significance of sOX40L in atherosclerosis.

Abstract:Aim To analyse the impact of metabolic syndrome(MS) on brachial-ankle pulse wave velocity (BaPWV) in a group of health examination people.Methods The medical examination and blood chemical analysis and related metabolic items were completed in these medical examination people.The brachial-ankle pulse wave velocity (BaPWV) was assessed by using a Form PWV device.Using the standard of MS,defined by the Chinese Diabetes Society in 2004,the people were grouped by the different Metabolic level.The BaPWV were compared among these groups, and the correlation between the BaPWV and the metabolic abnormality was analyse.Results The prevalence of MS was 13.26%,in which the metabolic abnormality in four items was 2.43%.With the increase of abnormal metabolic items and age,the BaPWV increased gradually(P<0.05) and were higher than that of the control group.The same tendency appeared in different sex groups also.Although the single metabolic items were abnormal,compared with the control group,the increase of the BaPWV was significant(P<0.05),especially in the hypertension group.The subjects were also classified into three groups based on the age(<40 years old group,40～59 years old group and>60 years old group).Besides the aged group(>60 years old group),the BaPWV increased with the increasing number of the metabolic abnormality.The correlation between the BaPWV and the metabolic abnormality was significant(left r = 0.42,P<0.01,rightr=0.45,P<0.01).Conclusion The BaPWV and the prevalence of the MS increased with age.All items of the metabolic abnormality were risk factors of the BaPWV increase.The more the metabolic abnormality items were,the more the BaPWV increased.

Abstract:Aim To explore the effect of probucol,rosuvastatin and their combination on atherosclerosis and cardiac function in essential hypertensive patients.Methods A total of 178 essential hypertensive patients,were randomly assigned to rosuvastatin group(rosuvastatin,10 mg/d),probucol group(probucol,500 mg/d) and the combination group(rosuvastatin+probucol).All the patients were treated and followed up for 8 months.The changes of matrix metalloproteinase(MMP),tumor necrosis factor(TNF-α),atherosclerosis plaque of artery and cardiac function between baseline and the end of 8 months were analysed.Results At the end of 8 months,rosuvastatin group,probucol group and the combination group showed reductions in MMP,TNF-α,the intima media thickness(IMT) of the common carotid artery and the size of plaque in coronary artery(CS);and the combination group showed more reductions compared with rosuvastatin group and probucol group.Left ventricular ejection fraction(LVEF) increased from 60.2%±11.0%to 64.4%±12.5%(P<0.05) and left ventricular peak filling rate(LVPFR) increased from 1.78±0.39 to 2.89±0.67(P<0. 01) in the combination group.Conclusion The combined treatment with probucol and rosuvastatin yielded better results in the context of improving left ventricular function and showed favorable effects on atherosclerosis plaque of artery.

Abstract:Aim To observe the clinical efficiency and safety of ozagrel sodium(OS) combined with Xueshuantong (XST) in treatment of patients with acute cerebral infraction(ACI).Methods 127 patients with acute cerebral infraction were randomly divided into control group,treatment group and combined-treatment group.The control group was given vinpocetine sodium chloride injection(VSCI).The treatment group was given OS.The combined-treatment group was given OS and XST(once a day for 14 days).All the patients were observed for the clinical efficiency and safety. Results Neurological function was significantly improved in the combined-treatment group as compared with the treated group and the control group after 7 days or 14 days of therapy(P<0.05).The total effective rate in combinedtreatment group was obviously higher than those in the treatment group and the control group(81.4%,54.7%and 52.4%,respectively P<0.05).No statistically significant difference was observed in the treatment group and the control group(P>0.05).No significant difference was observed in adverse effect in the three groups.Conclusion Ozagrel sodium combined with Xueshuantong in the treatment of patients with acute cerebral infraction is an effective and safe method.

Abstract:Aim To investigate the prognostic value of cardio-ankle vascular index(CAVI) in the occurrence of coronary heart disease(CHD).Methods 497 patients treated with coronary angiography were studied.All patients underwent cardio-ankle vascular index(CAVI) and serum factors evaluation in addition to history collection.The severity of coronary artery stenosis was evaluated by the number of diseased arteries and Gensini score.The risk factors of coronary heart disease and CAVI were compared between each group.The predicting role of these variables,especially CAVI,to the occurrence of CHD was analysed with Logistic regression.Results There were significant differences in sex composition,age,hypertension,diabetics,hyperlipidemia and cigarette smoking between the CHD group and the non-CHD group(P<0.05).The CAVI were significantly higher in patients with coronary artery disease than in the normal subjects(P<0.05).There was a positive linear trend between CAVI and Gensini's score(r=0.763,P<0.01). Multiple logistic analysis indicated that age,hypertension,diabetics,hyperlipidemia,cigarette smoking and CAVI were significantly correlated to the occurence of CHD(P<0.05).Conclusion Just like age,hypertension,diabetics,hyperlipidemia and cigarette smoking,CAVI were also independent risk factors of CHD.CAVI was an unattached predictor of the severity of coronary artery stenosis.

Abstract:Aim To evaluate left cardiac function and morphology in patients with ventricular septal defect (VSD) after transcatheter closure by echocardiography.Methods In 60 patients with transcatheter closure of VSD by Amplatzer occluder,echocardiography examinations were performed to calculate left ventricular end-diastolic diameter (LVEDD),left ventricular end-dlastolic volume(LVEDV) left ventricular end-systolic volume(LVESV),left atrium endsystolic diameter(LAESD),left ventricular ejection fraction(LVEF),systolic movement velocities(S'),early diastolic movement velocities(E') and late diastolic movement velocities(L') 1 day before,3 days,3 months and 6 months after the closure respectively.Results LVEDD,LVEDV,LVESV,LAESD,LVEF,S',E' and L' of 3 and 6 months after transcatheter closure were lower than those of 1 day before transcatheter closure and 3 days after transcatheter closure(P<0.05 or 0.01).There were no difference between 3 days after transcatheter closure and 1 day before transcatheter closure (P>0.05).Conclusion Transcatheter closure of VSD can improve left cardiac function and geometric morphology, at the same time it can eliminate the abnormal shunt.

Abstract:The virulence of a virus,perceived by the speading speed of the virus within a population,reflects the population' s genetic heterogeneity.When the difference of morbidity and mortality of a virus-caused disease within various ethnic groups is trivial,the virulence of the virus may be very powerful.In contrast,when the difference is significant, the virulence is quite low.The greater the difference between the ethnic groups,the lower the virulence.