Abstract:Aim To investigate the effect of 9-cis-retinoid acid (9-cisRA), a specific agonist of retinoid X receptors (RXR) on the differentiation of THP-1 human monocyte cell induced by phorbol-12-myristate-13-acetate (PMA). Methods Human monocyte cell line THP-1 was cultured and induced to macrophage by PMA treatment. The effect of 9-cisRA on the differentiation induced by PMA was studied. Cell morphology was observed by phase contrast microscope and the adhesion rate of THP-1 was detected by methyl thiazolyltetrazolium (MTT) assay. The cell surface markers involved in differentiation of monocyte/macrophage (CD11b, CD36) were analyzed by flow cytometry (FACS). Results The metamorphotic cells were decreased in 9-cisRA group. THP-1 cell adhesion rate was inhibited by 50% (P<0.01), and the cell surface markers (CD11b, CD36) were decreased by 50% and 28% (P<0.01) in 9-cisRA treated group. Conclusion The specific agonist of RXR, 9-cisRA could effectively inhibit the differentiation of THP-1 human monocyte induced by PMA.

Abstract:Aim To evaluate the effect of cariporide on the proliferation of rat aortic smooth muscle cells (VSMC) induced by advanced glycation end products (AGE). Methods The cultured vascular smooth muscle cells of rat (VSMC) were exposed to normal Dulbecco's Modified Eagle Medium (DMEM) or AGEs-supplemented DMEM for 24 hours in the presence or absence of cariporide. Proliferations of cells were observed by MTT colorimetric assay and determination of total protein level in cells. Intracellular pH and free Ca2+ concentration ([Ca2+] i) were measured by using the pH-sensitive fluorescent dye BCECF and the Fura-2 technique respectivelly. Results AGEs significantly increased the VSMC MTT light absorption (0.554±0.032 vs 0.155±0.018) and total protein level compared with the control group(193.3±10.7 μmol/L vs 127.8±8.2 μmol/L, P<0.01); cariporide significantly inhibited these effects induced by AGEs in a concentration-dependent manner. Cariporide 0.1, 1, 10 μmol/L reduced the MTT light absorption, from 0.554±0.032 to 0.402±0.028, 0.298±0.020, 0.174±0.019 while the total protein level of cells from (193.3±10.7) μmol/L to (180.9±9.8) μmol/L, (156.4±9.4) μmol/L, (130.6±8.8) μmol/L respectively. Moreover, cariporide simultaneously reduced the intracellular pH and Ca2+concentration. Conclusion AGE can significantly promote the VSMCs proliferation; cariporide can inhibit the VSMC proliferation induced by AGEs, the mechanism involves reducing the pHi and [Ca2+] i.

Abstract:Aim To study the effect of rhynchophylline and isorhynchophylline on the apoptosis of vascular smooth muscle cell (VSMC) induced by angiotensin Ⅱ (AngⅡ) its mechanism in rats. Methods The apoptosis model in VSMCs induced by angiotensin Ⅱ was established. The effect of rhynchophylline and isorhynchophylline on the cell morphology, apoptosis rate, [Ca2+]i concentration, Bcl-2 and Bax protein expression and mRNA expression were observed by invert phase contrast microscope, flow cytometry and RT-PCR. Results The rhynchophylline and isorhynchophylline could induce the apoptosis of VSMCs, increase cell apoptosis rate, decrease cell [Ca2+]i concentration and down regulate Bcl-2, Bax protein and mRNA expression. Conclusion The rhynchophylline and isorhynchophylline may induce the apoptosis of VSMC, whose mechanisms might be related to decreasing cell [Ca2+]i concentration and down regulating Bcl-2 and Bax protein expression and mRNA expression.

Abstract:Aim To produce specific monoclonal antibody (McAb) against human vascular endothelial cell membrane proteins for investigating the functions and relation to disease of vascular endothelial cells. Methods McAb was prepared with classical hybridoma technique. Human vascular endothelial cell line(ECV304)was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third days after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with cellular enzyme-linked immunoabsorbent assay (CELISA). Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using fast protein liquid chromatography(FPLC),and its Ig class, subclass and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Results One line of hybridoma cells with high expression of specific McAb termed as NH-2 against vascular endothelial cell membrane protein was obtained. The Ig subclass of the McAb NH-2 was IgG1 and the titer of ascitic McAb was 1×10-6. Furthermore, the content of ascitic McAb was 18.82 g/L. Flow cytometry, CELISA and western blot assays demonstrated that McAb NH-2 could specifically recognize antigen expressed on ECV304 and human umbilical vein endothelial cell(HUVEC). Meanwhile, the tissue specificity and cell specificity of antigen recognized by McAb NH-2 were identified better seprately by immunohistochemical ABC staining and flow cytometry. On the other hand, the molecular weight of antigen protein recognized by McAb NH-2 was about 40kD. Conclusions NH-2 can specifically recognize the natural antigen expressed mainly in vascular endothelial cells, which will potentially be usefull for investigation of the functions and clinic application values of vascular endothelial cells.

Abstract:Aim To study the mechanism of anti-injury of pariphyllin on the human umbiliar vein endothelial cell (hUVEC) ECV 304 induced by hydrogen peroxide (H2O2). Methods A model of endothelial cell oxidative damage induced into H2O2 was established, then cells were divided into five experimental groups including normal group, oxidative damage group and three different concertration pariphyllin groups. MTT assay was used to detect the protection of pariphyllin, reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the mRNA expression level of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), flow cytometry (FCM) was used to quantitate the expression of ICAM-1 and VCAM-1. Results Cell optical density in oxidation damaged group was lower than nomal control (P<0.01); when pretreated by the medicine, the OD value was increased, and it had no significant difference in the high density pariphyllin than normal control (P>0.05). The mRNA expression of ICAM-1and VCAM-1 was significantly descended (P<0.01) in medicine treatment groups than in H2O2 damage group, FCM result discovered that the numbers of masculine cells which express ICAM-1 or VCAM-1 were obviously more than other groups (P<0.01), but pretreated group could make the number lower and the effection was dose dependent. Conclusions Pariphyllin has the protective action on oxidative damage of ECV304, which was related to stabilizing the cell wall to inhibit the inflammatory reaction induced by ICAM-1 and VCAM-1, and then protect endothelial cell, prevent atherosclerosis.

Abstract:Aim To determine the content of apelin in acute myocardial ischemia and probe the protective mechanism of exogenous apelin-13 on myocardium ischemic injury in rats. Methods The content of apelin in myocardium and plasma was detected by enzyme linked immunosorbent assay (ELISA) in SD rats suffered from myocardial ischemia by ligating the left anterior descending coronary artery (LAD) for 30, 60 and 120 min respectively. Then the rats were randomly divided into sham operated group, model control group and two apelin-13 groups, to which apelin-13 of 10 and 100 μg/kg was infused via caudal vein 5 min before a 120 min-myocardial ischemia. The cardial function was measured by electrophysiolograph. Cardiomyocyte apoptosis was detected with TUNEL and Bcl-2, Bax and Caspase-3 protein expression of myocardium with Western blotting. Results The content of apelin in myocardium was markedly increased during myocardial ischemia 30 to 60 min and decreased to below basal level on ischemia 120 min (P<0.01). The content of apelin in plasma increased gradually during myocardial ischemia 30 to 120 min (P<0.01). In myocardial ischemia models induced by ligating LAD for 120 min, apelin-13 (100 μg/kg) infused pre-ischemia can significantly improve cardial function, inhibit the cardiomyocyte apoptosis (P<0.01), increase Bcl-2 protein expression and decrease Bax and Caspase-3 protein expression. Conclusion The content of apelin in myocardium was increased in a compensatory role in early myocardial ischemia and decreased in sustained myocardial ischemia. The protective mechanism of exogenous apelin-13 on ischemic myocardium may be related to inhibition of cardiomyocyte apoptosis.

Abstract:Aim To study the effect and possible mechanism of mangiferin on myocardial ischemia reperfusion (MIR) injury in rats. Methods Fouty-eight SD rats were randomly divided into six groups: sham group, model group, mangiferin groups of 10 mg/kg, 20 mg/kg, 40 mg/kg and Diaoxinxuekang group. Each group were injected with corresponding concentrations of mangferin, Diaoxin xuekang or equal volume of saline gastrogavaged for 21 d. 0ne hour after the last administration myocardial ischemia reperfusion models were obtained by ligated left anterior descending coronary artery 40 minutes and followed by 120 minutes reperfusion and sham group was given all the procedures except ligation. Ultrastructure of myocardium with TEM and infiltration of polymorpho nuclear neutrophils (PMN) in myocardium with myeloperoxidase (MPO) were observed; serum activity of lactate dehyd -rogease (LDH), myocardial activity of superox -ide dismutase (SOD) and contents of malondialdehyde (MDA) were detected respectively. Results Mangiferin could improve myocardial pathology and ultrastructurein mangiferin 10 mg/kg group the activities of SOD was higher (P<0.05); the contents of MPO, MDA, LDH were significantly lower (P<0.05). Conclusion Mangiferin can protect MIR. The myocardial protective mechanism of it may be realized by enhancing the activation of SOD activity,enhancing myocardial antioxygen capability, stabilizing myocardial cellular membrance and alleviating infiltration of polymerrphonuclear neutrophils(PMN) in myocardium.

Abstract:Aim To explore the effect of STIM1 knockdown on cell cycle of vascular smooth muscle cell. Methods Rat VSMC was isolated and primary cultured. Ad-si/rSTIM1 and Ad-hSTIM1 were transfected into VSMC. The protein of STIM1, p21 and pRb were measured by western blot, the cell cycle of VSMC was analyzed by flow cytometry. Results On 48 h after transfected Ad-si/rSTIM1, the expression of STIM1 protein was decreased significantly compared to that in Ad-hSTIM1-treated group (P<0.05). The percent of G0/G1 phase cell was significantly increased (P<0.05). The expression of p21 protein was increased and the expression of pRb protein was decreased (P<0.05). However, the cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 restored these responses. Conclusions STIM1 knockdown up-regulates the expression of p21 and down-regulates the expression of pRb, which inhibits cell to progress into S phase. Stim1 maybe have a key role in the vascular smooth muscle cell proliferation.

Abstract:Aim To analyse the relation between heparanase (HPA) and stablilization of athersclerotic plaques in human coronary arteries; and to investigate the role of HPA in creating vulenerable sites in atherosclerotic plaques. Methods Samples of coronary arteries were obtained at autopsies of 36 patients with acute coronary syndrome. Eighty randomly selected blocks were studied by immunohistochemistry using antibody against HPA. Computer-aid planimetor was used for quantitative analysis. Results In unstable plaques, percentage of immunoreactive areas for HPA was significantly higer than that in stable plaques. Conclusions HPA in coronary atherosclerotic plaque is an important factor in determining stablilization of the plaques.

Abstract:Aim To compare and investigate the different methods for establishing type 2 diabetic atherosclerosis rat models. Methods 5 Wistar rats were chosen control group and 15 GK rats were randomly divided into three groups: hypertriglyceride diet (12 weeks) group, hypercholesterol diet with vitamin D (12 weeks) group and hyperchlesterol diet with vitamin D (24 weeks) group. Histopathology was used to observe the atherosclerotic lesions. Results There were no obvious morphologic changes in these four groups by light microscope. The macrophage-related antigen expressions in the aorta were not observed in all the four groups. Absence in abnormal expression of SMC-related antigens was shown in all of them. Conclusion All of the three methods were not successful in establishing diabetic atherosclerotic rat models in Wistar rats.

Abstract:Aim To investigate the changes of serum soluble intercellular adhesion molecule-1 (sICAM-1) concentration among healthy smokers, transient ischemic attack (TIA) and cerebral infarct patients and to study the effects of smoking on the occurrence and development of cerebral infarct. Methods The level of serum sICAM-1 was measured by two-layer antibody sandwich enzyme-linked immunosorbent assay (ELISA) in 31 cases of smokers with cerebral infarct and 31 cases of non-smokers with cerebral infarct, 31 cases of smokers with TIA and 25 cases of non-smokers with TIA, 26 healthy smokers and 28 healthy non-smokers. The cerebral blood flow velocities were measured by transcranial doppler (TCD). Results The level of serum sICAM-1 in cases of smokers with cerebral infarct, TIA and healthy smokers were higher than that of non-smokers with cerebral infarct, TIA and healthy non-smokers respectively (P<0.05). The level of serum sICAM-1 was well correlated with smoking index (r=0.428,P<0.01). The blood flow velocity in bilateral MCA increased compared to that before smoking cigarette (P<0.05). Conclusion Smoking evaluates the sICAM-1 level of the health, transient ischemic attack and infarction patients. sICAM-1 is involved in the process of the occurrence and development of cerebral infarct.

Abstract:Aim To observe the effects of ulinastatin (UTI) on the level of matrix metalloproteinase-9 on plasma in patients receiving cardiac valve replacement therapy and to investigate the protective effects of Ulinastatin on lung injury. Methods 40 adult patients, ASA score Ⅱ～Ⅲ, scheduled for elective cardiac valvular replacement, were randomly allocated into two groups, Ulinastatin group (group U, n=20) and control group (group C, n=20). Group U in which patients received a total of 12 ku/kg of ulinastatin. 6 ku/kg ulinastatin was given a IV bolus on introduction of anesthesia and before CPB. Group C received an equal amount of isotonic sodium chloride solution. Blood samples were taken from radial artery before introduction of anesthesia, 10 min after initiation of CPB, 30 min after initiation of CPB, 1 h, 3 h and 6 h after CPB for determination of plasma matrix metalloproteinase-9 concentrations. VD/VT and alveolar-arterial oxygen A-aDO2 were checked before introduction of anesthesia, 1 h, 3 h, 6 h after initiation of CPB studied after CPB. Results There was no significant difference between two groups in age, weight, operation time, CPB time, and aortic cross-clamping time. Compared with those before introduction of anesthesia, the concentrations of matrix metalloproteinase-9 increased significantly at 10 min after initiation of CPB to 6 h after initiation of CPB in two groups (P<0.05). And no difference between two groups before introduction of anesthesia. The concentration of VD/VT at 1 h after initiation of CPB to 6 h after initiation of CPB increased significantly (P<0.05). The concentrations of MMP-9 at 10 min after initiation of CPB to 6 h after initiation of CPB, VD/VT A-aDO2 at 1 h after initiation of CPB to 6 h after initiation of CPB in ulinastatin group was lower than those in control group significantly (P<0.05). Conclusions Ulinastatin suppressed the excessive release of matrix metalloproteinase-9 and protected lung injury in patients undergoing cardiopulmonary bypass.

Abstract:Aim To study the levels of interleukin-17 (IL-17) and C-reactive protein (CRP) in plasma of aging coronary heart disease patients with depression. Methods The concentrations of IL-17 and CRP in plasma were examined by using enzym-linked immunosorbent assay in 68 aging coronary heart disease patients which were divided into two groups: depression group (n=38) and un-depression group (n=30). Results Concentrations of IL-17 and CRP were significantly higher in aging depression patients with coronary heart disease than those of the control group (341.72±134.28 ng/L vs 214.33±82.51 ng/L and 4.93±2.15 μg/L vs 1.93±1.25 μg/L, P<0.01). Levels of IL-17 and CRP in acute coronary syndrom were higher than that in stable angina patients (382.17±165.35 ng/L vs 305.47±119.75 ng/L and 5.96±2.31 μg/L vs 4.12±2.44 μg/L, P<0.01). Concentration of IL-17 in aging coronary heart disease patients with minor to moderate depression and major depression were significantly higher than those of non-depression patients (310.27±138.25 ng/L and 395.42±158.75 ng/L vs 214.33±82.51 ng/L, P<0.05), the concentrations of IL-17 major depression in aging coronary heart disease depression were higher than that of minor to moderate depression in aging patients with depression. The plasma levels of CRP in aging coronary heart disease patients with minor to moderate depression and major depression were increased as compared with the non-depression patients (4.35±2.01 ng/L and 5.84±1.87 ng/L vs 1.93±1.25 ng/L,P<0.05). The levels of major depression were increased as compared with minor to moderate depression patients. The levels of CRP were positively related with IL-17 in aging coronary heart disease patients with depression (r=0.802, P<0.05). The concentration of IL-17 and CRP were positively associated with the scores of depression (r=0.674, P<0.05; r=0.421,P<0.05). Conclusion Interleukin-17 and C-reactive protein play a role in the mechanism of aging coronary heart disease with depression.

Abstract:Aim To investigate the associations of serum interleukin-18 (IL-18) and C-reactive protein (CRP) with endothelial dysfunction in patients with essential hypertension. Methods Flow-mediated dilation (FMD) in right brachial artery was detected to reflect endothelial function by ultrasound. Serum IL-18 and CRP levels were also assayed. Bivariate correlation analysis was applied to show the correlation of serum IL-18 and CRP levels with FMD. Multiple linear regression was performed followed by unconditional logistic regression. Results The serum IL-18 and CRP levels were significantly higher in essential hypertension group than those in control group (P<0.01), but the FMD was significantly lower (P<0.01). In patients with essential hypertension, with the degree of blood pressure ascending, the serum IL-18 and CRP levels climbed gradually, but the FMD was descending (P<0.05). The serum IL-18 and CRP levels were significantly increased in FMD abnormal subgroup in comparison with those in FMD normal subgroup (P<0.01). Serum IL-18 and CRP levels were negatively correlated with FMD, respectively (P<0.01). In stepwise multiple linear regression model, after adjustion with other factors, the correlations of IL-18 and CRP levels with FMD persisted, respectively. The relationship between FMD and other study factors were analyzed by non-conditional logistic stepwise regression. Serum IL-18 and CRP levels were the important independent risk factors for FMD abnormal. Conclusions Serum IL-18 and CRP were negatively correlated with endothelial dysfunction in patients with essential hypertension, and the correlations persisted after adjustion with other factors, respectively. Serum IL-18 and CRP were the important independent risk factors for carotid artery plaque. The study suggests the link between inflammation and endothelial dysfunction in patients with essential hypertension.

Abstract:Aim To study the influence of immunological function on serum lipid level in patients with systemic lupus erythematosis (SLE). Methods Taking anticardiolipin antibody (ACL) and T-lymphocyte subsets counts as the starting points, the association between the immunological function and the serum lipid level including high density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC) and triglyceride (TG) levels were observed in 149 SLE inpatients. Results HDLC was significantly and negatively related to ACL (r=-0.448,P=0.005), while LDLC was positively related to C4 (r=0.427,P=0.007). Conclusion The aberrant humoral immunological functions in SLE patients may influence their serum lipid level.

Abstract:Aim To investigate the influence of ralated factors to different antihypertensive effective rate of metoprolol with the same genotypes of CYP2D6 and β1-adrenergic receptor (β1-AR). Methods 63 cases of 1 or 2 grade essential hypertension patients with the *10*10 of CYP2D6*10 genotype and Arg/Arg of β1-AR Gly389Arg genotype were chosen and administrated with metoprolol 100 mg/d. Indexes, such as blood pressure and heart rate, were collected every two weeks during the follow-up visit. After 8 weeks, the relationships between the related factors, such as age, baseline blood pressure, body mass index (BMI), hyperlipidemia and smoking, and antihypertensive effective rate of metoprolol were analysed. Results According to antihypertensive efficacy, 40 patients were effective to metoprolol, while 23 were ineffective. The effective rate was 63.5%. Monofactorial analysis indicated that patients with higher baseline systolic blood pressure and normal BMI had greater effective rate than the others (P<0.05). Furthermore, multifactoring regression analysis also claimed that baseline systolic blood pressure showed the significant effect to metoprolol. Conclusion Baseline blood pressure and BMI are related to the different antihypertensive effective rate of metoprolol.

Abstract:Aim To investigate the association between a 1562C→T mutation of matrix metalloproteinase-9 (MMP-9) gene and carotid artery arteriosclerosis in type 2 diabetes mellitus (T2DM) of Guilin. Methods A case-control study for 158 Guilin Han Chinese subjects (including 98 type 2 diabetes mellitus and 60 normal control) was performed. The number of the 1562 C→T mutation alleles were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results The frequencies of genotype and allele showed no significant difference between control groups and T2DM group, and the frequency of the T genotype and T allele at 1562 were significantly higher in T2DM with carotid artery arteriosclerosis group than in T2DM without carotid artery arteriosclerosis group and control subjects (P<0.05). The analysis showed that MMP-9 T allele was an important risk factor of carotid artery arteriosclerosis in T2DM(OR=2.160, 95%CI:1.026～4.547). Conclusion The T allele of C1562T polymorphism of MMP-9 gene is related to carotid artery arteriosclerosis in Guilin T2DM. The T allele may be a risk factor.

Abstract:Aim To explore the relationship of microalbuminuria and the coronary artery stenosis degree in essential hypertension patients. Methods According to the results of urine protein quantitative in 24 hours, 82 patients in essential hypertension patients were selected as microalbuminuria (MAU) positive group and 82 microalbuminuria negative patients were selected as control group. The severity of coronary artery stenosis was compared. Results Compared to the microalbuminuria negative group [42/82 (52%)], the morbidity of coronary heart disease (CHD) were higher in microalbuminuria positive group [60/82 (74%)], with more prevalence in two vessels disease group (25% vs 12%, P<0.05); and three vessels disease group (15% vs 3%, P<0.05). Conclusion The incidence and severity of coronary heart disease were much higher in essential hypertension patients associated with microalbuminuria.

Abstract:Aim To study the relationship between serum uric acid level and metabolic syndrome (MS) and its cardiovascular complication. Methods A total of 1000 subject with metabolic syndrome were enrolled in the study. MS was defined using NCEP-ATPⅢ criteria. The relationship between MS and its carvdiovascular complication and hyperuricemia were studied. Results The prevalence of hyperuricimia with MS was 53%. Uric acid level was the most significantly associated with blood pressure, triglycerides, waist circumference. Hyperuricemia was more common in men than in women, and was older than nohyperunicemia. Serum uric acid level was significantly associated with cardiovascular complication of MS. Conclusions The hyperuricermia was significantly associated with MS and its cardiovascular complication. The hyperuricermia may be an important part of MS and an important risk factor of cardiovascular diseases.