Abstract:The features of atherothrombotic diseases are the rupture of atherosclerotic plaques and thrombosis.Platelet activation and aggregation play a pivotal role in the pathogenesis and clinical manifestation.Similar to neuronal and immunological synapse,synaptic structure is also formed between platelets during thrombus formation.The microenvironment of platelet synapse facilitates signal transduction that has been accepted as contact-dependent events due to their occurrence after platelet contact begins,which is believed to play a role in the thrombus formation and stability.Axonal guidance molecules(eg Ephrins and Semaphorins) have been found in platlet synapse.In addition to axonal guidance,axonal guidance molecules have been shown to participate in the pathophysiological processes,including immune response,tumor metastasis,angiogenesis,and thrombosis. Most recently,it was found that deletion of a semaphorin family member,Sema4D,attenuated platelet hyperreactivity in the setting of dyslipidemia and protected from the development of atherosclerotic plaques.This article summarizes the progress in the understanding of the molecular mechanisms by which axonal guidance molecules affect atherothrombosis.

Abstract:Aim To observe the expression of calcium sensing receptor(CaSR) in atherosclerosis rat vascular endothelial cells and the relationship with apoptosis. Methods 144 Wistar rats were randomly divided into control group(n=72) and atherosclerosis groups(n=72). Early atherosclerosis models were copied by injection of vitamin D3 D360 million units/kg body weight,and then fed with high fat diet for 6 weeks. Serum triglyceride(TG) and total cholesterol(TC) were detected by automatic biochemistry analyzer. The morphological changes were observed under optical microscope. The mRNA and protein expressions of CaSR,Bcl-2,Bax and Caspase-3 were analyzed by Western blotting and RT-PCR. Apoptotic cells were measured by TUNEL staining. Results Compared with control group,the apoptosis index and the expression of CaSR,Bax and Caspase-3 were increased,Bcl-2 expression were decreased,the changes in aorta thoracica were aggravated in the atherosclerosis group. Conclusion CaSR may participate in the apoptosis of vascular endothelial cell,and then accelerate the formation of atherosclerosis.

Abstract:Aim To investigate the role and mechanism of TSG on the apoptosis of human umbilical vein endothelial cells(HUVEC) induced by H2O2. Methods HUVEC were treated with TSG(0.1,1,10 and 100 μmol/L) for 24 hours then exposed to H2O2(100,200,300,400 and 500 μmol/L) for 24 hours,the optimal concentration of H2O2 and TSG were dedected by MTT and Flow Cytometry. Morphology of apoptosis and the protective effect of TSG on HUVEC induced by H2O2 were observed by Hoechst33258 staining.The mRNA and protein expression of Caspase-3 were detected by RT-PCR and Western blot analysis respectively. Results According to the MTT and Flow Cytometry,the optimal concentration for H2O2 to establish apoptosis model and for TSG to protect HUVEC induced by H2O2 were 300 μmol/L and 10 μmol/L respectively.Compared with the control group,the group of 300 μmol/L H2O2 inhibited the cell proliferation,increased the number of apoptotic cells and the expression of Caspase-3 significantly.Compared with H2O2 group,10 μmol/L of TSG improved the rate of cell proliferation,inhibited cell apoptosis,and decreased expression of the caspase-3 significantly(P<0.05). Conclusion TSG could inhibit H2O2-induced apoptosis of HUVEC,and its mechanism was associated with the inhibition of Caspase-3 expression.

Abstract:Aim To investigate the protective effect of Astragali radix extract on vascular cell adhesion molecule-1 expression of mice vascular endothelial cell against tumor necrosis factor-α(TNF-α). Methods Adhesion model was established by THP-1 cells and mice endothelial cell in vitro.The cells were pretreated by different dose and different time of Astragali radix extract before induced by TNF-α,and the adhesion rate were detected.The levels of vascular endothelial cell adhesion molecule VCAM-1 in the cell culture were determined with ELISA.The expression of VCAM-1 and NF-κB subunit(p65) were evaluated by Western blot. Results The expression of VCAM-1 and NF-κB was increased obviously after induced by TNF-α;While the expression of VCAM-1 and the effect of NF-κB p65 protein nuclear translocation induced by TNF-α were inhibited after pretreatment of Astragali radix extract in a dose-and time-dependent manner.The reduction of adhension of monocytes to endothelial cells,the down-regulation of the expression of VCAM-1 and reduc-tion of the expression of NF-κB were apparent(P<0.05) at the concentration of 120 mg/L preincubated 4～8 h. Conclusion Astragali radix extract can inhibit the TNF-α-induced expression of VCAM-1 and reduce the adhension of monocytes,by which the damage to vascular endothelial cells was relieved.The mechanism may be related to the role of inhibiting the activation of NF-κB.

Abstract:Aim To investigate the effect of proprotein convertase subtilisin/kexin 9(PCSK9) siRNA on inflammatory factor expression in oxidized low density lipoprotein(ox-LDL) induced THP-1 macrophage cells. Methods The siRNAs for PCSK9 were designed and synthesized,then THP-1 macrophages were transfected with 80 nmol/L PCSK9 siRNA by positive ion liposome Lipofectamine 2000 for 24 h and then a high level of ox-LDL(80 mg/L) for an additional 24 h.Reverse transcription polymerase chain reaction(RT-PCR) was conducted to detect interleukin-1α(IL-1α) mRNA,interleukin-6(IL-6) mRNA and tumor necrosis factor-α(TNF-α) mRNA. Results Ox-LDL increased IL-1α mRNA,IL-6 mRNA and TNF-α mRNA expression.The expression of IL-1α mRNA,IL-6 mRNA and TNF-α mRNA in the macrophage pretreated with PCSK9 siRNA was decreased significantly,in comparison with ox-LDL-treated group(P<0.05). Conclusion PCSK9 siRNA can repress inflammation factor expression of THP-1 macrophage cells induced by ox-LDL.PCSK9 may be involved in adjustment for inflammation.

Abstract:Aim To investigate the effect of Shensongyangxin capsule on QT dispersion and connexin43 expression in heart failure rats. Methods Heart failure rats models were built by constricting abdominal aorta,and were lavaged with Shensongyangxin capsule for 8 weeks.Ventricular electrophysiology were measured by inserting home-made electrode into subcutaneous,left ventricular morphostructure,myocardial fibrosis,and connexin43 distribution were respectively observed by HE staining,Masson staining,immunohistochemical staining. Results Heart failure rats QT dispresion were significantly longer(37.20±9.94 ms,P<0.05),cardiomyocytes were misaligned,myocardial fibrosis area were significantly increased(101217.30±33970.02 μm2,P<0.05),and connexin43 distribution were significantly decreased(55.93±11.61,P<0.05).Shensongyangxin capsule can shorten QT dispresion(25.50±8.21 ms) of heart failure rats,increase connexin43 distribution(69.09±16.59) and decrease myocardial fibrosis area(13580.64±8213.73 μm2) in myocardium of heart failure rats. Conclusion Shensongyangxin capsule can shorten QT dispresion of heart failure rats,increase connexin43 distribution and decrease myocardial fibrosis area in myocardium of heart failure rats.

Abstract:Aim To investigate the effect of water extract propolis(WEP) on apoptosis in human umbilical arterial smooth muscle cell(HUASMC),and explore the mechanism of water extract propolis on inhibition of atherosclerosis and it's implications. Methods HUASMC were cultured and identified by immunocytochemistry technique.Cultured HUASMC were randomly divided into 5 groups: control group,model group and WEP groups(treated with 50 mg/L,100 mg/L and 200 mg/L WEP respectively).Intracellular total cholesterol(TC) and free cholesterol(FC) were measured by high performance liquid chromatogram,the content of cholesterol ester(CE) was obtained by subtracting the FC from TC.Cellular apoptosis index was tested by flow cytometry. Results The content of intracelluar CE in model group was more than that in control group(P<0.01).The content of intracellular CE in 50 mg/L,100 mg/L and 200 mg/L WEP groups were less than that in the model group(P<0.01),and with the increase of the concentration of WEP the content of intracellular CE showed the decreasing tendency.The cell apoptosis index in model group was higher than that in control group(P<0.01).The cell apoptosis index in 50 mg/L,100 mg/L and 200 mg/L WEP groups were lower than that in the model group(P<0.01),and with the increase of the concentration of WEP the cell apoptosis index showed the decreasing tendency.The content of intracellular CE was correlated with the cell apoptosis(r=0.964,P<0.01). Conclusion WEP can reduce the apoptosis of HUASMC induced by ox-LDL which may be concerned with the inhibination of intracellular CE accumulation.

Abstract:Aim To construct the soluble epoxide hydrolase(sEH) gene-specific recombinant vectors pSUPER retro neo,and selectively knockdown the expression of sEH in mice cardiomyocytes by RNA interference(RNAi),and to select the plasmids having the best silence effect to sEH. Methods Two pairs of siRNA that target at sEH gene were designed,and to construct the siRNA expression vectors specific to sEH gene(EH-1 and EH-2).There were plasmids carrying a nonspecific siRNA coding sequence(pCN) as the negative control group and the blank control group involved.Then the plasmids were transfected into primary cultured mice cardiomyocytes by FuGENE HD.The mRNA and protein expressions of sEH were analyzed by RT-PCR and Western Blotting respectively. Results The expressions of mRNA and protein of sEH in EH-2 group(0.202±0.017 and 0.212±0.029,P<0.01) were significantly decreased compared with that of blank control group,negative control group,and EH-1 group. Conclusion Recombinant expression vectors have been constructed,and RNA interference can selectively knockdown sEH expression in cultured cardiomyocytes.This lays a foundation for further research of RNAi on cardiomyocytes.

Abstract:Aim To observe the effect of flavonoids from Cuscuta Chinensis on myocardial cell apoptosis and the related gene expression in ischemic/reperfused myocardial infarction. Methods The left anterior descended coronary artery was ligated for 30 min and reperfused for 2 h to make ischemia-reperfusion injury model.50 SD rats were randomly divided into the sham operation group,ischemia-reperfusion group,the low and high dose groups of flavonoids from Cuscuta Chinensis and isosorbide dinitrate group.Creatine kinase(CK) and lactate dehydrogenase(LDH) were assayed.The apoptotic index(AI) was analyzed by TUNEL staining.The protein expression of Bcl-2 and Bax were measured by immunohistochemistry and Western Blotting in each group,the ratios of Bcl-2 and Bax were calculated. Results Compared with the sham operation group,the ischemia-reperfusion group of enzymes CK and LDH,AI,and Bcl-2 and Bax protein expression were significantly higher(P<0.01);The enzyme CK and LDH,and AI in the low and high dose group of flavonoids from Cuscuta Chinensis were decreased compared with those of the ischemia-reperfusion group(P<0.01).The expression of Bcl-2 was increased and the expression of Bax was decreased(P<0.01).The isosorbide dinitrate group showed no significant difference when compared with the high dose group of flavonoids from Cuscuta Chinensis(P>0.05). Conclusion The pretreatment of flavonoids from Cuscuta Chinensis could dose-dependently decrease enzymes CK,LDH levels,increase the expression of Bcl-2 protein and Bcl-2/Bax,reduce the expression of Bax protein,and inhibit the apoptosis of myocardial cell.The therapeutic effects was similar to isosorbide dinitrate.

Abstract:Aim To analyze the risk factor of asymmetric leukoaraiosis(LA) and explore its incidence and its pathogenesis. Methods 266 consecutive patients elder than 40 years were included prospectively.Their clinical data were analyzed respectively.All of them had accepted a magnetic resonance lmaging(MRI) and a Ultrasonic examination.The white matter hyperintensities(WMH) volume of fluid attenuated inversion recovery(FLAIR) were quantified with a semi-automated method,Leukoaraiosis patients with a ratio of WMH volumes in heavy side and in light side >1.5 were considered as asymmetric LA,and numbers of lacunes were counted. Local factors were records for the analysis. Results 32 cases of asymmetric leukoaraiosis were found,the incidence of asymmetric LA was 12.03%.Comparing the risk factor of asymmetric LA patients and symmetric LA,the two groups had significant difference in age,history of hypertension,diabetes,carotid artery plaques/stenosis,ischemic stroke,and the heavy side of asymmetric LA had more lacunar infaction,higher Crouse scale.Binary Logistic regression showed age,history of hypertension,lacunar infarction and carotid artery plaques/stenosis were included in the model. Conclusion The risk factors for asymmetric LA are age,history of hypertension,lacunar infarction and carotid artery plaques/stenosis.

Abstract:Aim To observe the effect of cyclosporine A on serum interferon-γ(IFN-γ),nitric oxide synthase(NOS) and nitric oxide(NO) in patients with chronic aplastic anemia(CAA). Methods 60 CAA patients were randomly divided into two groups.Control group was given Yixuesheng,while observation group was given cyclosporine A on the basis of control group.Two groups serum IFN-γ,NOS and NO before and after treatment were compared. Results Control groups serum IFN-γ(27.4±3.3 ng/L),NOS(30.8±4.0 kU/L) and NO(28.1±2.7 μmol/L)after treatment had no obvious change.Observation groups indexes(21.7±2.5 ng/L,22.3±3.6 kU/L和9.5±1.8 μmol/L)were obviously lower than before treatment and control group(P<0.05). Conclusion Cyclosporine A can decrease serum NO through inhibition of bone marrow failure,and thus reduce the damage degree of oxygen free radical and protect vascular wall in a certain degree.

Abstract:Clearance of apoptotic cells that have significant impacts on the composition,progression and stability of atherosclerotic is an important physiologic function of macrophage.This review will address the molecular and cellular mechanisms that are thought to govern macrophage recognizing,ingesting and swallowing apoptotic cells as well as the role of improving the stability of atherosclerosis.

Abstract:Deregulation of fatty acid synthase(FASN) catalyzed de novo fatty acids biogenesis could play a central role in the pathogenesis of atherosclerosis.We reviewed pharmacological and genetic alterations of FASN activity that have been shown to significantly influence artherosclerosis and its risk factors including obesity,type 2 diabetes.First,the endogenous fatty acids which are catalyzed by the key enzyme FASN are one of atherosclerotic plaque compositions.Secondly,FASN influences the oxidized low density lipoprotein intake and cholesterol efflux in macrophage,which would absolutely affect the plaque formation.Thirdly,FASN plays a key role in monocytes differentiation.Inhibitting FASN may reduce the formation of foam cells.In addition,FASN involved in lipid metabolism is also associated with metabolic diseases,such as obesity and diabetes which are the risk factors for coronary heart disease.We propose that the development or the progression of artherosclerosis can be prevented or reversed by the modulation of FASN status.The use of FASN inhibitors might be a valuable therapeutic approach for coronary disease.

Abstract:Aim To explore the changes in extracellar signal-regulated prote in kinase(ERK1 /2) in endothelial cell induced by Angiotensin Ⅱ at the different time courses,and its possible molecular mechanism.Method s Human umbilicalve in endothe-lial cell(HUVEC) were cultured in vitro and intervened by AngⅡ.HUVEC were divided into 2 groups,the control group,AngⅡ group(stimulated by AngⅡ10-6mol/L for 24h).Flow cytometery with Annexin V-FITC /PI double staining and Hoechst33258 fluo-rescence staining were used to detect apoptosis of HUVEC.The expressions of apoptosis-association genes Bcl-2,Bax were detected by RT-PCR and ERK1 /2 levels were detected by Western-blotting at different time points.Results 10-6mol/L Angiotensin Ⅱ stimulation stimulated cell apoptosis.Bcl-2mRNA levels were time-dependently decreased,the radio of Bcl-2 /Bax was decreased markedly(p<0.05).Phosphorylation of ERK1 /2 began to increase and reach the peak at 18 h(p<0.01).Conclusions Cell apoptosis is possibly important factor for atherosc lerosis.One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and the radio of Bcl-2 /Bax.There is a probability that activated ERK1/2 signal pathway is involved in the process of pathologic and physiologic reaction in the apoptosis of endothelial cell induced by Angiotensin Ⅱ.

Abstract:Objective TanshinoneⅡ-A(Tan),a bioactive diterpene isolated fromSalvia miltiorrhiza Bunge(Danshen),possesses anti-oxidant and anti-in-flammatory activities.The present study investigated whether Tan can reduce and stabilize atherosclerotic plaques in Apolipoprotein E knockout(ApoE-/-) mice maintained on a high cholesterol diet(HCD).Methods and Results Six week-old mice challenged with HCD were ran-domly assigned to 4 groups: C57BL/6J,ApoE-/-,ApoE-/-+30 mg/kg.d Tan and ApoE-/-+10 mg/kg.d Tan.After 16 weeks of inter-vention,Tan treated mice showed decreased atherosclerotic lesion size in the aortic sinus and face aorta.Furthermore,immunohistochemical a-nalysis revealed that Tan rendered the lesion composition a more stable phenotype as evidenced by reduced necrotic cores,decreased macrophageinfiltration,increased smooth muscle cell and collagen content.Tan also significantly reduced in situ superoxide anion production,aortic expres-sion of NF-κB,and matrix metalloproteinase-9(MMP-9).In vitro treatment of RAW264.7 macrophages with Tan significantly suppressed oxi-dized LDL-induced reactive oxygen species production,pro-inflammatory cytokine(IL-6,TNF-α,MCP-1) expression,and MMP-9 activity.Conclusions Tan attenuates the development of atherosclerotic lesions and promotes plaque stability in ApoE-/-mice by reducing vascular oxi-dative stress and inflammatory responses.Our findings highlightTan as a potential therapeutic agentto preventatherosclerotic cardiovascular dis-eases.

Abstract:Background and Aim Adiponectin(APN) is a potent cardioprotective molecule.The present study aims to investigate the under-lying mechanism(s) for its cardioprotective effect.Methods Primary cardiomyocytes were isolated from neonatal rats and an invitro model of hypoxia-reoxygenation(H/R) was established.The cardiomyocytes were randomly divided into six groups: salinegroup(control),dithiothreitol(DTT) group(5 mmol/L DTTfor 2 h),H/R group,H/R +APN group(incubation with 30 mg/LAPN,followed by H/R),H/R +APN +SB203580(SB) group(treatment with 30 mg/L APN and 5μmol/L SB,followed by H/R),and H/R +SB group(exposure to 5μmol/L SB and then H/R).Cell death was detected by measuring lactate dehydrogenase(LDH) release.The expression levels of hypoxia-inducible factor-1alpha(HIF-1α) and endoplasmic reticulum(ER) stress-relatedgenes including GRP78,caspase-12,C/EBP homologus protein(CHOP),and p38 mitogen-activated protein kinase(MAPK) wereexamined.Results Cardiomyocytes exposed to H/R showed a significant increase in LDH leakage and HIF-1αprotein levelscompared with the control cells(p<0.05).The H/R-provoked cell death was profoundly attenuated by the pretreatment with APNalone,SB alone,or both,which was coupled with decreased expression of GRP78,caspase-12,CHOP,and p38 MAPK.Conclu-sions These results provide new insights into the mechanism of APN-mediated cardioprotection,which may be partially due to inhibi-tion of ER stress response.

Abstract:<正>Reverse cholesterol transport(RCT)is assumed to play a critical role in the pathogenesis of atherosclerosis.Cellular cholesterol efflux,by which cholesterol is transported from peripheral cells to high-density lipoprotein(HDL)acceptor

Abstract:Background and Aim Asymmetric dimethylarginine(ADMA),an endogenous inhibitor of nitric oxide synthase(NOS),has been shown to be an independent predictor of coronary heart disease(CHD).Dimethylarginine dimethylaminohydrolase-2(DDAH2) promotes the metabolism of ADMA and plays a key role in formation of the atherosclerosis.We hypothesized that genetic variation inDDAH2 gene might alter the susceptibility to CHD.Methods We tested our hypothesis in a case-control studies.We used ahaplotype-tagging single nucleotide polymorphisms(SNP) approach to identify tag SNP in DDAH2.The SNP were genotyped by poly-merase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and ligase detection reaction(LDR)-sequencing in 1650patients with CHD and 1920 control subjects.Results Apromoter variant-449C/G(rs805305) and -1415G/A(rs2272592)in DDAH2 was identified in the region containing DDAH2.The frequency of those polymorphism were consistent with the lawof Har-dy-Weinberg.The frequency of rs805305 CG +GG or G allele was not significantly different between CHD and wild-type genotype(OR: 0.667,95%CI: 0.374 to 1.187,p>0.05).The frequency of rs2272592 GA +AA or A allele also showed no significant difference between CHD and wild-type genotype(OR: 1.420,95%CI: 0.899 to 2.242,p>0.05).No association was observed be-tween the DDAH2 variant and CHD.These results was independent of age,gender,hypertension,diabetes and hyperlipidemia.Conclusions Our results suggest that although DDAH2/ADMA pathway acts as a critical regulator of coronary atherosclerotic heartdisease,the DDAH2 common variant may not predict the susceptibility to CHD in Chinese population.

Abstract:Aim To explore the mechanisms of microRNA 200a in hepatic insulin resistance induced by interleukin-6(IL-6),specifically to in-vestigate the role of microRNA 200a in hepatic glucogen and insulin signal pathway transduction.Methods The cell model forhepatic insulin resistance was induced by IL-6(10μg/L) for 24 hours in NCTC1469 cell.The level of glucogen was reduced.The expression of microRNA 200a was tested by q-PCR.The level of glucogen was quantified in NCTC 1469 transfected with microRNA200a mimics.The target gene of microRNA200a was predicted by bio-information software.The protein level of the target gene wastested by Western Blotting.Results The level of glucogen and the insulin signal pathway was inhibited in NCTC 1469 cells trea-ted by IL-6.The expression of microRNA 200a was decreased in NCTC 1469 cell induced by IL-6.The level of glucogen and theinsulin signal pathway were stimulated in NCTC 1469 cell transfected by microRNA 200a mimics.The protein level of Pten was de-creased by overexpression of microRNA200a.Conclusions IL-6 downregulates the expression of microRNA200a in hepatic insu-lin resistant NCTC 1469 cells.The microRNA 200a stimulates biosynthesis of glucogen as well as insulin signal pathway accompanied by down-regulating the expression of Pten.

Abstract:Aim Hydrogen(dihydrogen,H2) is an effective antioxidant to reduce oxidative stress and oxidative stress is implicated in atherogene-sis.In this study we examined whether hydrogen-saturated saline can prevent atherosclerosis in apolipoprotein E knockout(apoE-/-) mice fed either chowdiet or high-fat diet,and characterized the underlying molecular mechanisms.Methods and Results The atherosclerotic lesion formation displayed by oil red O staining positive area was reduced significantly in either aortic root section or aortic arch en face in hydrogen administrated apoE-/-mice fed either chowdiet or high-fat diet,compared to the control.Plasma analysis by enzymatic method showed that total cholesterol(TC) and non-high-density lipoprotein cholesterol(non-HDL-C)were remarkably decreased by treatment with hydrogen.Western blot analysis revealed a significant decrease of both plasma apoli-poprotein B(apoB) level and hepatic expression of apoB after hydrogen treatment,suggesting hydrogen could downregulate the expression of the major protein constituent of non-HDL.In addition,spectrophotometric measurement showed that plasma levels of malondi-aldehyde(MDA) and serum amyloid Awas decreased and paraoxonase-1 activity was increased in mice treated with hydrogen,suggesting plasma lipid oxidation and peroxidation was impaired by hydrogen treatment.Besides,the MDA content of the non-HDL,whichseparated by ultracentrifugation from the plasma of mice treated with and without hydrogen,was reduced by hydrogen,suggesting the oxidation of non-HDL was impaired by hydrogen.Moreover,we found hydrogen treatment significantly suppressed the production of tumor necrosis factor-α(TNF-α) and interleukin-6 in RAW264.7 macrophages after stimulation with the isolated non-HDL,suggesting hydrogen reduces atherogenesis by inhibiting non-high-density lipoprotein(HDL)-mediated inflammation.Furthermore,immunohis-tochemistry of aortic valve sections revealed that hydrogen attenuated lesion formation by suppressing the expression of several proin-flammatory factors and decreasing vessel wall infiltration of macrophages,indicating hydrogen-treatment reduces arterial inflammation.Besides,real-time PCR and western blot analysis disclosed that the expression of several transporter genes involved in the process ofreverse cholesterol transport,including hepatic scavenger receptor class B type I(SR-BI),ATP-binding cassette(ABC) transporters ABCG8,ABCB4,ABCB11,and macrophage SR-BI,were all induced by hydrogen treatment.Conclusion These results re-vealed that administration of hydrogen-rich saline reduces atherogenesis in apoE-/-mice fed a high-fat diet by inhibiting the non-HDL-mediated arterial inflammation and promoting the expression of genes involving reverse cholesterol transport.

Abstract:Aim To study the effect of interleukin-10(IL-10) on the expression of adhesion molecules and scavenger receptor lectin-like oxidized low density lipoprotein receptor-1(LOX-1) in human vascular endothelial cells,and to approach the function of IL-10 on anti-atherosclerosis. Methods Human umbilical vein endothelial cells were randomly divided into four groups: treated with or without oxidized low density lipoprotein(ox-LDL),treated with IL-10 plus or minus ox-LDL for 24 h.Flow cytometry technique was used to detect the expressions of E-selectin and intercellular adhesion molecule-1(ICAM-1) on the surface of the cells.Real-time PCR and Western Blotting analysis were used to detect mRNA and protein expression levels of LOX-1 in the cells. Results After 24 h stimulation,the expression level of E-selectin and LOX-1 on the surface of cells were significantly increased by 20% and 25% respectively in the ox-LDL treated group compared with untreated group.However these remarkable increments were revoked when IL-10 was present at the same time with ox-LDL.But,IL-10 alone had no such effect.The expression levels of ICAM-1 on the surface of cell was not affected by ox-LDL. Conclusion IL-10 has a protective effect on endothelial cells injury induced by ox-LDL,which may be one of the mechanisms of IL-10 in anti-atherosclerosis.The protective effect of IL-10 is probably by down-regulating the expression of LOX-1 and E-selectin on the surface of endothelial cells and inhibiting the adherence of monocytes to endothelial cells,reducing the cell activation.

Abstract:Aim To investigate the effects of chronic unpredictable mild stress(CUMS) on platelet(PLT)5-hydroxy tryptamine(5-HT) in rat fed with high fat diet(HFD) Methods 48 male SD rats were randomly and averagely divided into four groups: control group,CUMS group,HFD group and HFD+CUMS group,with 12 rats in each group.The rats in control group and CUMS group were fed with basic food and were once intraperitoneally injected with sodium chloride,and then fed with sodium chloride by intragastric administration once every day.The rats in HFD and HFD+CUMS group were fed with high fat diet and were once intraperitoneally injected with VitD3 600000 U/kg,and then fed with propylthiouracil by intragastric administration once every day.The rats in CUMS and HFD+CUMS group were given CUMS from the 3rd week to 9th week.At the end of 9th week,platelet 5-HT in the serum were analyzed. Results The platelet 5-HT of the rats was 0.272±0.332 μmol/109 PLT,0.137±0.184 μmol /109 PLT,0.502±0.409 μmol /109 PLT,0.187±0.216 μmol/109 PLT in control group,CUMS group,HFD group,HFD+CUMS group respectively.Factor analysis showed that HFD had no significant effect on platelet 5-HT(P=0.177),while CUMS significantly decreased the concentration of platelet 5-HT(P=0.035),there was no interaction between HFD and CUMS administration on platelet 5-HT level(P=0.382). Conclusion CUMS decreased platelet 5-HT level in serum.But there was no interaction between HFD and CUMS on platelet 5-HT level in serum.

Abstract:Aim To investigate the effects of advanced glycation end products(AGE) on the expression of monocyte chemoattractant protein-1(MCP-1) and vascular cell adhesion molecule-1(VCAM-1) in human umbilical vein endothelial cell(HUVEC)and the intervention effect of atorvastatin. Methods Collagenase was used to isolate the endothelial cell from human umbilical vein;HUVEC were identified by immunocellochemistry and morphology;RT-PCR was performed to detect MCP-1,VCAM-1 and recepter for AGE(RAGE) mRNA expression;Reactive oxygen species(ROS) detectionkit was used to examine the level of ROS in HUVEC and inversion fluoescence microscope was used to observe the ROS level. Results The cultured cells were oval or polygon and retained cobblestone appearance through inverted phase contrast microscope,brown particles could be observed in cytoplasm(CD31 or CD34 positive cells) after immunocytochemical staining with CD31 or CD34;In comparison with control group,AGE(10-4~10-1 g/L)promoted MCP-1 and VCAM-1 mRNA expression in a concentration-dependent manner in HUVEC.The expression of MCP-1 and VCAM-1 mRNA was significantly elevated by AGE(10-4 g/L) compared with the control group(0.26±0.02 vs 0.17±0.04;0.22±0.02 vs 0.08±0.01,P<0.01);Compared with AGE group,atorvastatin(0.1,1,10 μmol/L) diminished MCP-1 and VCAM-1 mRNA expression induced by AGE in HUVEC in a concentration-dependent manner;Atorvastatin in a dose of 1 μmol/L,could significantly decrease the expression of MCP-1 and VCAM-1 mRNA induced by AGE(0.63±0.11 vs 1.03±0.07;0.21±0.03 vs 0.83±0.10,P<0.01);The level of ROS in AGE group was higher than that in control group,atorvastatin could obviously decline the ROS level induced by AGE in HUVEC.Furthermore,atorvastatin(0.1 μmol/L) was able to decrease RAGE mRNA expression remarkly induced by AGE in HUVEC(0.63±0.05 vs 1.19±0.12,P<0.01). Conclusion AGE could significantly increase MCP-1 and VCAM-1 mRNA expression in HUVEC;Atorvastatin could decrease the oxidative stress and inflammation gene expression induced by AGE in HUVEC through inhibiting the expression of RAGE.

Abstract:Aim To construct pEGFP-GPR109A recombinant plasmid by inserting GPR109A gene into pEGFP-N3 vector and establish a Chinese hamster ovary(CHO)cell line stably expressing nicotinic acid receptor GPR109A. Methods The pEGFP-GPR109A recombinant plasmid was constructed,which was subsequently transformed into DH5α E coli.After identification by PCR,digestion with restriction endonuclease and sequencing,the recombinant plasmid was transfected into CHO cells via lipofectamine 2000.The stable transfectants were screened by antibiotic G418.The fluorescent signal of cloned cell lines were detected by fluorescent microscope.The GPR109A gene mRNA expression was analyzed by RT-PCR and the fusion protein expression of green fluorescence protein and nicotinic acid receptor GPR109A(GFP-GPR109A) was detected by Western Blotting.The cell localization of fusion protein was detected by laser scanning confocal microscope. Results The results of PCR,restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pEGFP-GPR109A.Western Blotting showed that the fusion protein GFP-GPR109A was expressed stably in CHO cells.The fusion protein was mainly localized on cell membrane detected by laser scanning confocal microscope. Conclusion The eukaryotic expression vector pEGFP-GPR109A has been successfully constructed and a CHO cell line stably expressing fusion protein GFP-GPR109A was established,which facilitated the physiological and pathological function research of GPR109A and also provided important foundation for following up drug screening for atherosclerosis treatment.

Abstract:Aim To study the cardioprotective mechanism of hydrogen sulfide(H2S) on oxidative stress induced by hydrogen peroxide. Methods Primary-cultured cardiomyocytes were obtained from neonatal rats,an oxidative stress injury model was established by exposing the cells to H2O2 for 2 h.To study the cardioprotective mechanism of hydrogen sulfide(H2S) on oxidative stress induced by hydrogen peroxide,the cells were pre-incubated with ly294002 for 30 min.The morphology change of cardiomyocytes was observed by inverted phase contrast microscope,the activities of lactate dehydrogenase(LDH) and superoxide dismutase(SOD),and content malondialdehyde(MDA) were determined,the activation of the PI3K/Akt signal pathway was observed by Western blotting,the cardiomyocytes apoptosis was detected by AnnexinV/PI flow cytometry. Results After exposing to H2O2 for 2 h,the release of LDH,MDA and apoptosis were increased,the activities of SOD and ratio of Bcl-2/Bad were decreased(P<0.05).After pretreated with sodium hydrosulfide(NaHS) for 30 mins,the injury effect was attenuated,moreover,the phosphorylation of Akt,Bad and ratio of Bcl-2/Bad were upregulated.LY294002 could block the protection effect of NaHS partly. Conclusion H2S can attenuate hydrogen peroxide-induced rat neonatal cardiomyocytes injury,which may involve the PI3K/Akt pathway.

Abstract:Aim To investigate whether hypoxia could induce the expression of ABCA1 and cholesterol efflux in RAW264.7 cells. Methods RAW264.7 cells were cultured under hypoxia condition(37℃,5%CO2,1%O2)and normoxia conditon(37℃,5%CO2,20%O2) for 0,6,12,24 or 48 hours respectively,and ABCA1 mRNA and protein expression was detected by real-time PCR and Western blot,respectively.Using ox-LDL 3H-cholesterol,ApoA-I-mediated cholesterol efflux in RAW264.7 cells under hypoxia condition,was examined by scintillation counting. Results Expression of ABCA1 was down-regulated in RAW264.7 cells after being exposed to hypoxia conditon.Meanwhile,cholesterol efflux mediated by ApoA-I decreased after hypoxia treatment. Conclusion Hypoxia could significantly inhibit ABCA1 expression and cholesterol efflux mediated by ApoA-I in RAW264.7 cells.The inhibitory effect of hypoxia on cholesterol efflux may closely correlate with the development of atherosclerosis.

Abstract:Aim To investigate the effects of arecoline on blood coagulation and hemorrheology in type 2 diabetic rats. Methods Sprague-Dawley(S-D) rats were fed high-fat and high-fructose diet for four weeks and then were injected streptozotocin(30 mg/kg) to induce the type 2 diabetes.Once the type 2 diabetes models were set successfully,type 2 diabetic rats were randomized into five groups: normal control group,diabetes models group,arecoline groups(1 mg/kg,5 mg/kg and 20 mg/kg).The rats were treated with arecoline through intragastric administration for twelve weeks.At the end of the twelfth week,the level of fasting blood glucose,the level of blood plasma fibrinogen(Fbg) and the coagulation indexes including thrombin time(TT),prothrombin time(PT) and activated partial thromboplastin time(APTT) were detected.The hemorrheology indexes inculding blood viscosity,erythrocyte sedimentation rate,volume of packed red cells,erythrocyte aggregation index and erythrocyte deformity index were also detected. Results Compared with the normal control group,the level of fasting blood glucose was significantly increased,TT,PT and APTT were significantly decreased and the level of blood plasma Fbg was significantly increased in the diabetes models group.Compared with the normal control group,blood viscosity,erythrocyte sedimentation rate,volume of packed red cells and erythrocyte aggregation index were significantly increased,but erythrocyte deformity index was significantly decreased in the diabetes models group.Compared with the diabetes model group,the level of fasting blood glucose was significantly decreased and the indexes of coagulation and hemorrheology were improved in arecoline(5 mg/kg and 20 mg/kg) group.Conclusion Arecoline improves the blood coagulation and hemorrheology in type 2 diabetic rats.

Abstract:Aim To screen differential microRNA(miRNA) expression profiles of CD4+T lymphocyte from the patients with unstable angina pectoris(UAP) and the healthy controls by microarray analysis technique.To elucidate the mechanism responsible for modulation of CD4+T lymphocyte and provide insights into the effects of miRNA on UAP. Methods The CD4+T lymphocyte were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human peripheral blood by magnetic cell sorting system(MACS).The miRNA expressions profiles of CD4+T lymphocyte were screened using oligonucleotide microarray and differentially expressed miRNA were identified by using microarray analysis software.The microarray results were confirmed by real-time quantitative polymerase chain reaction(qRT-PCR). Results The expression of miR-155,miR-21,miR-424 and miR-127-3p were up-regulated over 1.5 folds,and the expression of miR-30b and miR-181a were down-regulated over 0.5 folds. The qRT-PCR results were in accordance with the microarray outcomes. Conclusion The differentially expressed miRNA of CD4+T lymphocyte may participate in the occurring and developing of UAP.