Abstract:Aim To discuss the dynamic changes and significance of the compensatory angiogenesis and related gene expression in muscle after limb ischemia.Methods Femoral artery ligation method was used to establish nude mice limb ischemia model.3 days,1,2,3 and 4 weeks after limb ischemia,nude mice limb ischemic changes were observed respectively.Hematoxylin-Eosine staining and CD34 immunohistochemical staining were used to observe ischemic morphological changes in muscle tissue.The application of Western Blotting and reverse transcription polymerase chain reaction detection of hypoxia inducible factor 1α(HIF-1α),hepatocyte growth factor(HGF) and vascular endothelial growth factor(VEGF) was used to observe the dynamic changes of gene expression in ischemic muscle.Results Nude mice limb gangrene was the most serious in 1～2 weeks after ischemia and improved 3～4 weeks after.After the limb ischemia,muscle fibers shrink and deformation in 3～4 weeks were getting better.The microvascular count was the highest in 2 weeks after limb ischemia.The gene expression of HIF-1α and HGF was the strongest in 3 days after limb ischemia,VEGF gene expression reached a peak at 1 week end.In 3～4 weeks after limb ischemia,all the gene expression were close to normal.Conclusion The short-term process of angiogenesis was mediated by HIF-1α,HGF and VEGF gene expression after the limb ischemia,but a limited number of capillary formation was not enough to compensate the state of limb ischemia.

Abstract:Aim To explore the effect of fenofibrate(Fen) and pioglitazone(Pio) on arteriae aorta remodeling in the fructose-induced metabolic syndromic(MS) Rats.Methods SD rats breeded with high fructose fed to raise MS rats,and Fen or Pio were used to intervent MS rats singly and jointly.The differences in arteriae aorta morphosis of MS rats were analyzed and compared among groups with different intervents.Results The arteriae aorta remodeling were amended in MS rats intervened by Fen and Pio.Relative to MS rats,rats intervened with Fen or Pio singly exhibited a more smoothing aortic tunica intima,less vascular medio-smooth muscle proliferation,and thinner vascular wall.Pio-intervention even displayed a more regular array of medio-smooth muscle cell and elastic fibers.Combination with fenofibrate and pioglitazone intervented MS rats,which built a more normal arteriae aorta in morphosis.Conclusions Intervention with fenofibrate or pioglitazone singly can restrain arteriae aorta pathologic remodeling in MS rats.The effectiveness of pioglitazone is more significant than that of fenofibrate.Synergistic effect of fenofibrate and pioglitazone can further improve and even reverse the vascular remodeling in MS rats by different mechanisms and acting targets.

Abstract:Aim To investigate whether estrogen deficiency may impact the platelet aggregation function in female SD rats and the underlying mechanisms relative to nitric oxide(NO).Methods Female SD rats were randomly divided into three groups: sham,ovariectomy,and ovariectomy supplemented with estrogen group.Each of them received sham operation,ovariectomy or ovariectomy followed by estrogen supplementation respectively.Estrogen supplementation started 3 days after the ovariectomy and continued for the following 4 weeks,then the animals were sacrificed.The serum was obtained to perform the estrogen assay.The platelet-rich plasma was collected to measure its platelet aggregation ability,and to detect plasma cGMP(Guanosine 3,5-cyclic monophosphate),the NO bioactive index,via the method of ELISA.Results The serum estrogen level in ovariectomized female SD rats was much lower than that in shamed animals,estrogen supplementation effectively restored the ovariectomized animals' serum estrogen.The platelets aggregation in ovariectomized rats was remarkably higher than that in shamed animals,estrogen supplementation could lower the platelets aggregation ability,though not to the sham level.The plasma cGMP level in the ovariectomized rats was significantly lower than that of shamed animals,estrogen supplementation may remarkably enhance the plasma cGMP,to a level in keeping with the sham group.Conclusion Lack of estrogen may boost the platelets aggregation in female SD rats,the mechanism might be relative to the down-regulation of NO/cGMP by estrogen depletion.

Abstract:Aim To explore whether homocysteine thiolactone(HTL)-induced vascular endothelial dysfunctions relate to activation of peroxisome proliferator activated receptor-γ(PPARγ).Methods The endothelium-dependent relaxation(EDR) and non-endothelium-dependent relaxation(n-EDR) of aortic rings in rats induced by acetylcholine and sodium nitroprusside(SNP) were examined respectively.The various biochemical parameters in vascular tissues or blood sera were also analyzed.Results Incubating HTL(10 mmol/L) with aortic rings for 30 min significantly inhibited EDR of isolated aortic rings,reduced nitro oxide(NO) content,increased activity of superoxide dismutase(SOD) and content of malondialdehyd(MDA) in vitro(P<0.01).The incubation of rosiglitazone(1 mmol/L) with aortic rings for 30 minutes prior to adding HTL significantly lessened HTL induced-injuries of EDR,preserved NO content,inhibited elevation of SOD activity and MDA content in aortic tissues(P<0.05 or P<0.01).Incubation of Captopril(0.03 mmol/L) and Apocynin(0.03 mmol/L) with aortic rings for 30 minutes prior to adding HTL respectively also protected the vascular endothelium from injury and the change of biochemistry parameters in aortic tissues(P<0.05 or P<0.01) in vitro.Rats were administered HTL(50 mg/(kg·d)) by gavage for 8 weeks that resulted in an inhibition of EDR of aortic rings,increases of MDA content,reduction of both paraoxonase-1(PON1) activity and NO content(P<0.01),but had no effect on activity of total nitric oxide synthase(TNOS),activity of endothelial nitric oxide synthase(eNOS),activity of induced nitric oxide synthase(iNOS) in sera and activity of erythrocytic SOD(P>0.05).The treatment of rosiglitazone(10,20 or 40 mg/(kg·d)) by gavage prior to gavage of HTL dose-dependently improved EDR of aortic rings injured by HTL in vivo,inhibited elevation of MDA content,recovered PON1 activity and NO content in sera induced by HTL(P<0.05 or P<0.01).The treatment of capropril(20 mg/(kg·d)) and apocynin(200 mg/(kg·d)) also significantly reduced injury of EDR and the changes of biochemistry parameters in sera(P<0.05 or P<0.01).The treatment of HTL in both vivo and vitro had no effect on the non-endothelium-dependent relaxation by SNP(P>0.05).Conclusion HTL-induced vascular endothelial dysfunction may relate to oxidative stress and involve in inhibiting the activation of PPARγ or down-regulating the expression of PPARγ.

Abstract:Aim To investigate the effects of adiponectin on endoplasmic reticulum stress injury of the 3～4 days SD rat cardiomyocytes,which was induced by the intervention of H2O2.Methods Primary cardiomyocytes were obtained from neonatal rat and cultured by enzymatic digestion methods.The morphology of neonatal rat cardiomyocytes was studied by phase contramicroscope.Its molecular markers were observed by α-actin immunocytochemistry.Primary 3～4 days cells were used in experiment,and they were randomly divided into control group,H2O2 group(200 μmol/L H2O2,2 h),H2O2+10 mg/L APN group,H2O2+20 mg/L APN group,and H2O2+30 mg/L APN group.The change of morphology of cardiomyocytes was observed by electron microscope.The content of lactate dehydrogenase(LDH) was detected by chemistry chromatometry;the cardiomycocytes apoptosis was detected by agarose gel electrophoresislevels and Annexin V-FITC/PI flow cytometry.The expressions of GRP78 and caspase-12 were detected by Western Blotting and RT-PCR.Results Adiponectin pretreatment significantly decreased the apoptosis rate(43.6%±3.8% vs 70.7%±6.4%),and the release of LDH(686.7±61.1 U/L vs 1411.5±189.7 U/L,P<0.05).The expression of GRP78 and caspase-12 attenuated(mRNA: 0.56±0.03 vs 1.25±0.50;0.83±0.04 vs 1.32±0.15),(protein: 0.66±0.03 vs 0.92±0.50;0.64±0.03 vs 1.24±0.50).Conclusion Oxidative stress enhances the expressions of GRP78 and caspase-12,and makes endoplasmic reticulum stress to start.Adiponectin which can attenuate endoplasmic reticulum stress,have a protective effect on myocardial cells.

Abstract:Aim To investigate the effects of baicalin on the expression of toll-like receptor 4(TLR4),inhibitor of κB(I-κB),and the production of tumor necrosis factor-α(TNF-α) in macrophage cells RAW264.7 induced by lipopolysaccharide(LPS).Methods RAW264.7 cells were cultured in vitro,and treated with different concentration of baicalin in the presence or absence of LPS.The mRNA and protein expression of TLR4 were determined by reverse transcription PCR and Western Blot,respectively.The expression of I-κB was detected by Western Blot.The concentrations of TNF-α in the supernatant was determined by enzyme linked immunosorbnent assay.Results LPS significantly up-regulated the expression of TLR4 and production of TNF-α in macrophage cells,and markedly inhibited the expression of I-κB.Pretreated with baicalin down-regulated LPS-induced TLR4 expression in both mRNA and protein levels,as well reduced the secretion of TNF-α.Baicalin significantly inhibited the degradation of I-κB.Conclusion Baicalin might effectively down-regulate TNF-α production in LPS-induced macrophage cells through inhibiting the expression of TLR4 and degradation of I-κB.The antiinflammation effects may be associated with the amelioration of atherosclerosis damage by baicalin.

Abstract:Aim To investigate the effects of advanced glycation end products(AGE) and metformin on activity and expression of endothelial nitric oxide synthase(eNOS) in cultured human umbilical vein endothelial cells(HUVEC).Methods HUVEC were co-incubated with different concentrations of AGE-BSA and metformin for 3,6,12 and 24 hours respectively.HUVEC viability was measured by CCK-8;nitric oxide(NO) was measured by the technique of nitrate reductase;the activity of NOS was determined by the spectrophotography and protein expression of eNOS was measured by Western Blotting.Results AGE-BSA inhibited the proliferation of HUVEC,while metformin promoted the proliferation of HUVEC.AGE-BSA significantly inhibited the generation of NO and the activity of NOS in a concentration and time-dependent manner(P<0.01).Metformin(compared with the control group) or with AGE-BSA co-interventions(compared with the AGE group) significantly increased the generation of NO and the activity of NOS(P<0.01).The expression of eNOS had a significant reduction when HUVEC were incubated with AGE-BSA for 24 hours,while the expression of eNOS had a significant increase when incubated with metformin.Compared with AGE-BSA group,the expression of eNOS up-regulated in metformin intervention group(P<0.01).Conclusion Metformin ameliorates the dysfunction of HUVEC induced by AGE.

Abstract:Aim To investigate the activity and expression of calcineurin(CaN) in the apoptosis of rat vascular smooth muscle cells(VSMC) under the stimulation of interferon gamma(IFN-γ)and CD40 ligand(CD40L),and the relationship between CaN and apoptosis of VSMC.Methods VSMC were obtained from SD rat and cultured in conditioned medium.Expression of CaN was measured by Western Blot and expression of CD40 and apoptosis of VSMC were examined by flow cytometry.Results The expression of CD40 in VSMC was clearly demonstrated.Activiation of VSMC by IFN-γ resulted in significant increase of expression of CD40 in VSMC(18.5%±0.2% vs 44.2%±2.4%).VSMC of control group showed low CaN activity(1.56±0.53 mmolPi/(g.h)),which was significantly enhanced by stimulation of IFN-γ,CD40L or combined stimulation of IFN-γ and CD40L respectively(6.48±1.09 mmolPi/(g.h),6.26±1.47 mmolPi/(g.h),11.70±2.73 mmolPi/(g.h),P<0.01),and so was the apoptosis of VSMC(P<0.01).The activity of CaN was significantly decreased after addition of FK506(2.74±0.30 mmolPi/(g.h),P<0.01).Conclusion The expression of CD40 was increased by the stimulation of IFN-γ.And combination of CD40-CD40L can increase the activity of CaN to induce the apoptosis of VSMC.

Abstract:Aim To observe the impact of oxidized low density lipoprotein(ox-LDL) on the expression of interleukin-6(IL-6) in rat vascular smooth muscle cells(VSMC) and investigate the role of toll like receptor 4(TLR4)/mitogen-activated protein kinase(MAPK)signaling pathway in this process.Methods VSMC were incubated with different concentrations of ox-LDL,the expressions of TLR4 mRNA and IL-6 mRNA were determined by reverse transcription-polymerase chain reaction(RT-PCR).The level of IL-6 in supernatant medium was detected by enzyme linked immuno sorbent assay(ELISA).Involvement of TLR4 for the phosphorylations of ERK1/2,P38 and JNK was shown using specific blocking anti-TLR4 antibody and the protein expressions were determined by Western blot.To confirm the involvement of TLR4/MAPK signaling pathway in the expression of IL-6 induced by ox-LDL,VSMC were pretreated with PD98059,SB203580,SP600125 and anti-TLR4 antibody respectively.Results After incubated with different concentrations of ox-LDL,the expressions of TLR4mRNA and IL-6 of VSMC were significantly up-regulated in a concentration-dependent manner(P<0.05).The phosphorylations of ERK1/2,P38 and JNK induced by ox-LDL were dramatically suppressed in the presence of anti-TLR4 antibody(P<0.01).The expression of IL-6 was attenuated by pretreated with PD98059,SB203580 and anti-TLR4 antibody prior to ox-LDL exposure(P<0.01),while SP600125 did not have the effect.Conclusion Our results demonstrate that ox-LDL can up-regulate the expression of IL-6 and the activation of TLR4 signaling in VSMC induced by ox-LDL may partly mediate the up-regulation of IL-6 via activation of ERK1/2 and P38.

Abstract:Aim To observe the effects of bezafibrate on the expression of caveolin-1 and the proliferation of vascular smooth muscle cells(VSMC) induced by platelet-derived growth factor(PDGF) and investigate the mechanism of it.Methods VSMC were cultured in vitro,the proliferation of VSMC were obtained through thiazolyl blue colorimetric technique(MTT).The expression of caveolin-1 was assayed by Western Blotting.Results Compared with the control group,PDGF could increasingly induce the proliferation of VSMC(P<0.01).Bezafibrate inhibited the proliferation of VSMC induced by PDGF in concentration-dependent manner(P<0.01).Compared with the control group,PDGF could decrease caveolin-1 protein expression(P<0.01).Bezafibrate increase the expression of caveolin-1 compared with the PDGF group(P<0.01).With the increase of Bezafibrate's concentration,the expression of caveolin-1 could increase accordingly.Conclusion Bezafibrate can decrease the proliferation of VSMC induced by PDGF and increase the expression of caveolin-1 protein.

Abstract:Aim To study the relationship between brachial-ankle pulse wave velocity(BaPWV) and serum malonyldialdehyde(MDA),superoxide dismutase(SOD) and nitric oxide(NO) in patients with hypertension co-existed with type 2 diabetes mellitus,and to investigate the effects of oxidative stress and vascular endothelial function on the development of arteriosclerosis.Methods 168 patients were devidid into normal control(n=40),hypertension(n=70) and hypertension complicated with type 2 diabetes mellitus group(n=58).The serum NO,MDA and SOD were measured by colorimetric method respectively,and BaPWV were recorded by an automatic arterial stiffness measurement device.Results Compared with the control group,serum MDA,BaPWV were significantly higher and SOD,NO was lower(P<0.05) in the hypertension group and the hypertension complicated with type 2 diabetes mellitus group.Compared with the hypertension group,serum MDA,BaPWV were significantly higher and SOD,NO was lower(P<0.05) in the hypertension complicated with type 2 diabetes mellitus group.Correlation analysis showed that BaPWV was positively correlated with age,systolic blood pressure,dilated blood pressure,cholesterol,fasting blood glucose(FBG),high-sensitivity C-reactive protein(hs-CRP),serum MDA(r=0.418,0.672,0.469,0.179,0.392,0.277,0.571,all P<0.05),and was negatively correlated with serum SOD and NO(r=-0.438,-0.571,all P<0.05).The analysis of multiple stepwise regression showed that BaPWV was positively correlated with systolic blood pressure,MDA,FBG and age.Conclusion Arterial elasticity significantly decreased and was correlated with increase of oxidative stress and reduction of vascular endothelial function in patients with hypertension complicated with type 2 diabetes mellitus,suggesting that oxidative stress and endothelial function plays an important role in the development of arteriosclerosis.

Abstract:Aim To study the influence of probucol on the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway of peripheral blood mononuclear cells in type 2 diabetic mellitus patients with coronary heart disease,so as to further investigate the anti-atherosclerosis mechanism of probucol.Methods 85 patients with type 2 diabetes and carotid atherosclerosis were divided into two groups: probucol treatment group(n=43),and control group(n=42).The treatment group were treated with probucol 0.375 g,2 times a day for 24 weeks.The effect of probucol on TLR4,NF-κB expression of peripheral blood monouclear cells was detected by Real-time PCR and Western blotting analysis before and after probucol treatment.The expression of interleukin-1β(IL-β) was detected by ELISA.Results Probucol obviously decreased levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDLC) and oxidized low density lipoprotein(ox-LDL) in plasma of type 2 diabetes.The expressions of TLR4,NF-κB and IL-β of peripheral blood mononuclear cells were significantly lower in probucol treatment group than that in control(P<0.05).Conclusions Probucol can not only regulate serum lipids,but also has the action of anti-atherosclerosis through inhibiting TLR4/NF-κB expression,which indicate that probucol might have the preventive and treatment role in patients with type 2 diabetes and atherosclerosis.

Abstract:Aim To observe the association between CXCL16 A181V polymorphism and the susceptiblity of acute coronary syndrome(ACS),furthermore,to approch the influence of CXCL16 A181V polymorphism to coronary severity in Xi'an Han population.Methods In the present study,we enrolled 183 patients with ACS and 88 patients without coronary artery disease(CAD).A181V polymorphism genotypes was determined by polymerase chain reaction-ligase detection reaction(PCR-LDR),and the coronary severity was counted with Gensini score.Results The comparative risk of ACS in AA homozygote was 4.053 times of VV homozygote,and the comparative risk of ACS in A allele carrier was 1.811 times to V carrier.The difference of Gensini score among A181 genotypes was significant(P=0.002).Conclusion A181V polymorphism is associated with susceptibility of ACS in Xi'an Han population,and also is associated with coronary severity.

Abstract:Aim To investigate the association between the polymorphisms of angiotensinogen gene(AGT235),angiotensin converting enzyme gene(ACEI/D),atrial natriuretic peptide gene(ANP2238) and essential hypertension in a southern Chinese Han population.Methods Performed by gene chip technology,the genotypes of 81 essential hypertensive people and 120 control people were measured to analyze the polymorphisms of AGT235,ACEI/D and ANP2238.Results A significantly higher frequency of ACEI/D DD genotype was observed in hypertensive group(48.1% vs 4.2%,P<0.001) than in control group.In hypertensive group,the frequency of AGT235 TT genotype and ANP2238CC genotype was respectively 53.1% and 6.1%,while it was respectively 45.8% and 8.3% in control group.No significant differences were observed(P>0.05).Conclusion The polymorphism of ACEI/D gene is related to essential hypertension.

Abstract:Aim To observe high-sensitive C-reactive protein(hs-CRP) expression in carotid atherosclerosis.Methods All enrolled patients were evaluated for carotid atherosclerosis by ultrasonography and detected for the level of hs-CRP in blood plasma.The hs-CRP content in carotid atherosclerosis group was compared with control group.The relationship between hs-CRP and carotid interia-media thickness(IMT)/carotid plaque scores was analysed.Results The level of hs-CRP(4.40 mg/L,0.20~16.91 mg/L) in carotid atherosclerosis group was higher than that in control group(1.07 mg/L,0.20~13.00 mg/L)(P<0.05).Partial correlation analysis of hs-CRP and carotid intima-media thickness/plaque scores showed positive correlation,and the partial coefficients were 0.4807 and 0.3024 separately(P<0.05).Conclusion The hs-CRP level is higher in carotid atherosclerosis group compared with that in control patients.The level of hs-CRP is positively correlated with the severity of carotid atherosclerosis.

Abstract:Aim To investigate the association of the complement factor H(CFH) 1277T>C polymorphism and unstable angina pectoris(UAP).Methods Frequency distribution of the genotypes and alleles of 1277T>C single nucleotide polymorphisms(SNP) of CFH were detected by using polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP) method in 150 cases with UAP and 146 normal controls of Hunan Han.Results Obvious frequency distribution difference of TT,TC genotypes and T,C alleles in CFH 1277T>C was observed between the UAP and the control groups(P<0.05),and the difference of the frequency of the CFH 1227C allele carriers(TC+CC genotypes) between the two groups was statistically significant(OR was 3.06,95%CI was 1.60~5.86,P<0.01).Conclusion It is suggested that CFH 1277T>C SNP is significantly associated with UAP in Hunan Han population.

Abstract:Aim To investigate the relation between the resistin gene 5'promoter region-420 C>G polymorphism and the age at onset of hypertension.Methods 121 hypertension were selected,including 43 age <40 years,78 age ≥ 40 years and 92 control,including 42 age <40 years,50 age ≥ 40 years,the resistin 420 C/G polymorphism genotype was analyzed by polymerase chain reaction-restriction fragment polymorphism(PCR-RFLP).Results The frequency of the G/G genotype of resistin single nucleotide polymorphism at-420 in younger hypertension was higher(P=0.006)and related to the age of onset.In young group,the frequency of the G/G genotype in hypertension was higher than control(P=0.001)and related to the onset of hypertension.Conclusion Younger-onset hypertension was related to the G/G genotype of resistin single nucleotide polymorphism at-420.

Abstract:Aim To discuss the relevance between cerebral infarction and carotid atherosclerotic plaque.Methods 47 patients with cerebral infarction were tested by ultrasound,and the site,nature and artery lumen diameter of their plaque were observed and compared with the result detected from 39 patients suffering carotid artery atherosclerotic plaque of the comparative group.Results The carotid artery atherosclerotic plaque of patients with cerebral infarction are mostly unstable plaques and associated with lipid levels(P<0.05),the distribution of carotid atherosclerotic plaque are mostly at the BIF.Conclusions The occurrence of cerebral infarction is closely related to carotid atherosclerotic plaque,the nature and distribution of carotid atherosclerotic plaque has something to do with the lipid levels.Through ultrasonic inspection we can detect the formation of plaque at an early stage,which is critical to the prevention of the occurrence of cerebral infarction.

Abstract:Aim To observe the changes in carotid arteriosclerosis plaque formation and injury between External Counterpulsation(ECP) group and no ECP group.Methods 165 patients(1-9 years) in treatment group by ECP and 68 patients without treatment were checked in carotid arteries with echo Doppler examination.Results Vessel wall injury in ECP group was 15.2% and carotid arteriosclerosis plaque formation was 11.5%.Vessel wall injury in no ECP group was 63.2% and carotid arteriosclerosis plaque formation was 51.5%.There were significant difference between the two groups(P<0.01).Conclusion ECP can reduce the incidence of vessel wall injury,preventing plaque formation.