Effects of bortezomib-based therapy on hemostasis system and circulating microparticles in multiple myeloma patients

AIM：To investigate the changes of hemostasis, thrombosis and total microparticles (TMPs) in multiple myeloma (MM) patients receiving bortezomib-based induction therapy (bortezomib+adriamycin+dexamethasone, PAD). METHODS：The levels of TMPs were detected by flow cytometry in 38 newly diagnosed MM patients and 30 healthy people. The changes of the platelet, coagulation, anticoagulation, fibrinolytic activation and TMPs in the MM patients before and after PAD teatment were also studied.RESULTS：Before treatment, the values of FVIII:C and vWF:Rco in MM patient were elevated ［(152.89±31.14)% and (165.69±38.43)%］, the activation of platelet aggregation was inhibited ［(63.76±21.36)%］, and the PAI level increased ［3.98(1.63)U/mL］. Compared with the healthy people, higher concentration of TMPs was observed in MM patients ［(640.65±214.22)/μL vs (134.29±63.09)/μL, P<0.01］, and the level of TMPs was positively correlated with serum β2-MG (r=0.672, P<0.01).After PAD therapy, platelet aggregation activation was restored ［(77.83±15.62)%, P=0.01］, and PAI level decreased ［0.88(1.38)U/mL, P<0.01］. The level of TMPs also decreased, after 3 cycles of PAD, to the value of (184.25±93.35)/μL. CONCLUSION：MM patients were characterized by impaired platelet, coagulation and fibrinolysis functions, and increased level of TMPs. PAD restored platelet function, decreased the levels of PAI and TMPs, which might partially explain the low incidence of thrombosis in the MM patients receiving bortezomib-based treatment.     

Protective effect of ethyl pyruvate on brain tissues in neonatal rats with hypoxic-ischemic brain damage

AIM：To study the effects of ethyl pyruvate (EP) on brain tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD) and its underlying mechanisms. METHODS：A total of 165 seven-day-old Sprague-Dawley (SD) rats were randomly divided into 3 groups：sham operation group (n=43), HIBD group (n=61) and HIBD+EP group (n=61). The rats in HIBD+EP group were intraperitoneally injected with EP (50 mg/kg) 30 min before operation, and once a day after surgery. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in brain homogenate, water content of brain and apoptotic cells in cortex were detected 3 days later. Ischemic and non-ischemic brain tissues were weighed to assess the extent of brain atrophy 14 days later. RESULTS：Higher level of SOD ［(125.78±18.35)×103 U/(g protein)］ and lower level of MDA ［(4.42±1.04) μmol/(g protein)］ in HIBD+EP group than that in HIBD group ［(97.84±15.50)×103 U/(g protein) and (6.02±0.89) μmol/(g protein), respectively］ was observed (P<0.05)．In addition, the water content of ischemic hemisphere was significantly higher than that of non-ischemic one in HIBD group (P<0.05), and was indistinguishable from that of non-ischemic one after EP treatment (P>0.05), indicating the protective effect of EP against brain edema. The apoptotic cells in cortex and hippocampus in HIBD+EP group ［(96.63±10.08)/field and (41.91±9.96)/field, respectively］ were obviously decreased compared with HIBD group ［(111.54±1.64)/field and (51.73±1.77)/field, respectively］, but still higher than those in sham operation group (P<0.05). The atrophy ratio of ischemic hemisphere in HIBD+EP group was (13.25±5.19)%, significantly lower than that in HIBD group ［(20.32±5.10)%, P<0.05］. CONCLUSION：Ethyl pyruvate is neuroprotective against HIBD in neonatal rats via increasing SOD level, decreasing MDA level, attenuating brain edema, decreasing apoptotic cells in cortex and alleviating atrophy of hypoxic-ischemic hemisphere.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Changes of mitochondrial peripheral-type benzodiazepine receptor during rat live regeneration

AIM: To investigate the expression profile of peripheral-type benzodiazepine receptor (PBR) involved in mitochondrial permeability transition (PT) regulation, and to observe the binding dynamic of the mitochondrial PBR with specificity ligand during rat live regeneration. METHODS: Liver regeneration model was produced by 70% partial hepatectomy (PH) performed in male SD rats. The animals of sham groups underwent the same surgical operations as PH groups did, but the liver lobes were not resected. The animals in the PH groups and corresponding sham groups were sacrificed at 3, 6, 12, 24, 48, 72, 120 and 168 hours after the operation. The livers were removed, weighted and processed for isolation of mitochondria. Semi-quantitative RT-PCR was performed to examine the expression level of PBR in 70% hepatectomized rat livers during the whole regeneration process and compared to that in the sham and normal groups. Compared with healthy rats, the kinetic parameters of PBR was evaluated by using a specific radioligand ［3H］-PK11195. RESULTS: Compared with healthy rats, the expression of PBR was unchanged. Meanwhile, the results obtained in the present experiments by scatchard analysis, Bmax of PK11195 for PBR significantly decreased, returned to normal level in 168 h after PH. Kd of PK11195 for PBR significantly decreased at 72 h and 168 h after PH of rat liver regeneration (P<0.01). CONCLUSION: The mRNA expression and evaluation of kinetic parameters of PBR may be related to the time-phase change of mitochondrial PT during rat liver regeneration after partial hepatectomy.     

Effects of salvianolic acid B on the energy metabolism and hydrocephalus in mice with cerebral ischemia

AIM: To explore the effects of salvianolic acid B (SalB) on the energy metabolism and hydrocephalus in mice with cerebral ischemia.METHODS: NIH mice were randomly divided into four groups: sham-operated group,cerebral ischemia group,SalB-treated group and nimodipine-treated group.The brain tissue energy charge (EC),phosphocreatine (PCr),the activity of ATPase,excitability amino acid (EAA) content and water content of brain were measured when cerebral ischemia for 30 min.RESULTS: EC (0.520±0.034),PCr content ［(98.344±13.249) μmol/g］,the activity of Na⁺-K⁺-ATPase ［(0.593±0.013)×10³ U/g］ and Ca²⁺-ATPase ［(0.484±0.053)×10³ U/g］ in SalB-treated group were significantly higher than those in cerebral ischemia group {EC (0.465±0.037),PCr content ［(81.614±9.919) μmol/g］ ,the activity of Na⁺-K⁺-ATPase ［(0.244±0.065)×10³ U/g］,the activity of Ca²⁺-ATPase ［(0.321±0.086)×10³ U/g］} (P<0.01).The glutamate (Glu) content ［(0.405±0.110) μmol/g］,aspartate (Asp) content ［(0.141±0.020) μmol/g］ and water content of brain ［(38.1±0.1)%］ in SalB-treated group were markedly lower than those in cerebral ischemia group ［ Glu content (0.550±0.140) μmol/g,Asp content (0.287±0.050) μmol/g,water content of brain (44.1±0.1)%］ (P<0.05,P<0.01).CONCLUSION: The increase in cerebral energy metabolism and the activity of ATPase,and decrease in EAA content in brain tissue are the mechanism of SalB alleviating hydrocephalus at the early stage of cerebral ischemia in mice.     

Changes in platelet-activating factor receptor binding characteristics in cerebral thrombotic core and penumbra of tree shrews

AIM: To observe the changes in platelet-activating factor (PAF) receptor binding characteristics and explore the action of PAF on formation of thrombotic core and penumbra following local cerebral ischemia. METHODS: Neuron's membrane protein was abstracted, and the local cerebral ischemia model were induced by photochemistry in tree shrews. The PAF binding sites on central neuron membrane were studied by-PAF binding assay. RESULTS: There were two different affinities of PAF receptors on tree shrew's brain cell membrane, with kD₁=(3.61 ±0.72) nmol/L and kD₂=(17.04±2.41) nmol/L, corresponding respectively to maximum number of binding sites: B_max1=(1 457.94±168.01) pmol/g protein and B_max2=(5 017.40±742.16) pmol/g protein. The binding sites decreased in ischemic core, penumbra and contralateral regions at 4,24 and 72 h after ischemia (P<0.01), with those of 24 h reaching the minimum levels. CONCLUSION: PAF receptors play an important role in cerebral ischemia, may be related to the secondary damage in ischemic penumbra, and also are molecular bases of brain injury induced by PAF.     

Endothelin receptor antagonist BQ123 inhibits calcification in vascular smooth muscle cells induced by β-glycerophosphate

AIM:To observe the effect of endothelin receptor antagonist (BQ123) on calcification in rat vascular smooth muscle cells (VSMCs) in vitro. METHODS:Calcification of cultured rat VSMCs was produced by incubation with β-glycerophosphate. Calcium content, Ca2+ deposition and alkaline phosphatase activity were analyzed to estimate the extent of calcification. The DNA synthesis was detected by ［3H］ -TdR and ［3H］-Leu incorporation. Osteopontin (OPN) mRNA was measured by competitive quantitative RT-PCR. Content of ET was measured by radioimmunoassay (RIA). RESULTS:The results showed that compared with the control, the content of calcium, ［45Ca2+］ uptake and alkaline phosphatases activities in calcified VSMCs increased by 118%, 174% and 7-fold (all P<0.01), respectively. The expression of OPN mRNA in calcified VSMCs was up-regulated by 86% (P<0.01). The calcified VSMCs grew rapidly, in which ［3H］-TdR and ［3H］-Leu were elevated by 71% and 35%. The content of ET in calcified VSMCs medium was increased by 35% as compared with control. Furthermore, calcified VSMCs plus BQ123 groups obviously relieved degree of calcification, of which calcium content, Ca2+ deposition and alkaline phosphatase activities were 33%, 37%, 40% lower than those in calcified VSMCs (P<0.01), respectively. The expression of OPN mRNA was down-regulated by 25% (P<0.01) and significantly inhibited VSMCs proliferation. CONCLUSION:BQ123 reduces VSMCs calcification, suggesting that ET promotes calcification in VSMCs mainly by ET/ ETA receptor pathway.     

Aldolase A level in malignant pleural effusion and its effects on proliferation,migration and invasion of human lung adenocarcinoma cells

AIM：
To investigate the levels of aldolase A (ALDOA), carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) in malignant pleural effusion (MPE) from patients with lung cancer and tuberculous pleural effusion (TBPE) from patients with tuberculous pleurisy, and to explore the effects of ALDOA on the proliferation, migration and invasion of human lung adenocarcinoma A549 cells. METHODS：Pleural effusion samples including 65 cases of MPE and 35 cases of TBPE were collected, and the levels of ALDOA, CEA and LDH were detected by ELISA and chemiluminescence assay. After A549 cells were treated with different concentrations of ALDOA, the proliferation, migration and invasion of the cells were investigated by MTT assay, scratch test, Matrigel assay and Transwell invasion assay. RESULTS：The levels of ALDOA, CEA and LDH in MPE were (46.8±21.4) μg/L, (82.2±56.6) μg/L and (755.8±382.5) U/L, respectively, which were significantly higher than those in TBPE ［(23.9±17.2) μg/L, (12.6±9.7) μg/L and (388.4±163.9) U/L, respectively; P<0.01］. The concentration of ALDOA in MPE from adenocarcinoma patients ［(71.7±32.1) μg/L］ was significantly higher than that in MPE from squamous-cell carcinoma patients ［(21.3±14.6) μg/L, P<0.05］. The concentrations of ALDOA in MPE and TBPE were positively correlated with the concentrations of CEA and LDH (P<0.01 or P<0.05). ALDOA enhanced the proliferation, migration and invasion of A549 cells in a concentration-dependent manner. CONCLUSION：The expression level of ALDOA in MPE is significantly higher than that in TBPE, especially in MPE from lung adenocarcinoma patients. There are highly positive correlations between ALDOA and CEA, ALDOA and LDH in pleural effusion. ALDOA concentration-dependently promotes the proliferation, migration and invasion of A549 cells.     

Effect of rosiglitazone on myocardial energy substrate utilization in type 2 diabetic rats

AIM: To study the effect of rosiglitazone (RSG) to improve insulin sensitivity on myocardial energy substrate utilization as well as the cardiac function in a rat model of type 2 diabetes mellitus. METHODS: Sprague-Dawley rats were conducted into three groups: chow-fed rats were fed with normal chow (12% of calories as fat); fat-fed/STZ rats were fed with high-fat diet (40% of calories as fat) for 4 weeks and then injected with streptozotocin 35 mg/kg intraperitoneal; fat-fed/STZ/RSG rats were fat-fed/STZ rats treated with rosiglitazone (3 mg·kg-1·d-1) for 2 weeks. A cannula connected to a passive transducer was inserted the heart for the measurement of the cardiac function including heart rate (HR), left ventricular end-diastolic pressure (EDP) and ±dp/dtmax. Then the isolated hearts were mounted onto a Langendorff perfusion apparatus to perfuse with Krebs-Henseleit buffer in the presence of 5 mmol/L glucose and 0.4 mmol/L ［3H］ labelled palmitate. Glucose uptake and ［3H2O］ collection were measured to evaluate the rate of carbohydrate and fatty acid oxidation. RESULTS: Compared with the chow-fed rats, fat-fed/STZ rats had a significantly depression of glucose uptake in the hearts ［(54.7±6.2 vs 69.0±5.7) μmol·g-1 dry weight, P<0.01］ after 30 min perfusion. The oxidation of glucose and palmitate were 18% and 82%, respectively. Paralleling the reduction was a change of EDP ［(14.3±1.8 vs 10.5±1.1) mmHg, P<0.05］ and -dp/dt ［(550±57 vs 650±42) mmHg/s, P<0.01］, indicating a impaired left ventricular diastolic function. In the hearts subjected to fat-fed/STZ group, rosiglitazone treated for 2 weeks resulted in a elevated level of glucose uptake ［(63.5±6.4 vs 54.7±6.2) μmol·g-1 dry weight, P<0.05］. A protective role of the ventricular function ［EDP decreased from （14.8±1.9） to （11.0±0.8） mmHg/s and -dp/dtmax increased from （558±60） to （629±51） mmHg/s, P<0.05］ were observed. CONCLUSIONS: Our study indicates that there is a depression of glucose oxidation and at increase in fatty acid oxidation in type 2 diabetic hearts. Elevation of insulin sensitivity using rosiglitazone increases the myocardial glucose metabolism and shows a benefitial result to heart functions.     

Effects of stretch on transient outward potassium and inward rectifier potassium current in cultured neonatal rat atrial myocytes

AIM：To investigate the effects of mechanical stretch on transient outward potassium current (Ito), inward rectifier potassium current (IK1) and action potential duration(APD) of cultured neonatal rat atrial myocytes. METHODS：Neonatal rat atrial myocytes were isolated and cultured on silicone sheeting with or without stretch for 24 h. The silicone membrane area was increased by 12% in stretched group. The cells without stretch served as control. Ito, IK1 and APD were recorded by the technique of whole-cell patch clamp. RESULTS：Compared with control group, Ito density in stretched myocytes was significantly reduced ［(16±04) pA/pF vs (121±29) pA/pF, P<001］, whereas IK1 density was increased ［(-108±08) pA/pF vs (-88±09) pA/pF, P<001］. The APDs at 50% and 90% levels of repolarization (APD50 and APD90) in the stretched cells were obviously decreased than those in non-stretched cells ［(105±14) ms vs (155±24) ms, (300±28) ms vs (563±36) ms, P<001］. CONCLUSION：Stretch stimulation leads to the reduction of Ito density, the increase in IK1 density and the shortness of APD in cultured rat atrial neonatal myocytes, which may contribute to atrial electrical remodeling induced by pressure overload.     

Correlation of serum uric acid level with carotid plaques and arterial stiffness in patients with essential hypertension

AIM：To study the correlation of serum uric acid (UA) level with carotid plaques and arterial stiffness in the patients with essential hypertension (EH), and to explore the predictive value of serum UA for evaluating EH preclinically. METHODS：A total of 92 patients with EH and 30 healthy individuals were enrolled. The value of UA and other indicators were detected. B-mode ultrasound examination was performed to measure the common carotid artery intima-media thickness (IMT) and the sites of plaque in the internal carotid-artery, external carotid artery and carotid bifurcations. Carotid-femoral arterial pulse wave velocity (CFPWV) was assessed by Complior atherosclerosis measurement instrument. RESULTS：The serum level of UA in the patients with EH was higher than that in control group ［(361.51±83.81） μmol/L vs (317.03±62.22) μmol/L, P<0.05］. The mean value and abnormal rate of IMT between hypertension group and control group were significant difference ［(0.69±0.14) mm vs (0.60±0.12) mm, 42.39% vs 10.00%, P<0.05］. In 92 EH patients, 45 cases had carotid plaques. These 45 cases were divided into 3 groups according to the plaque severity, among which the serum UA level had statistically significant differences ［(285.25±78.41） μmol/L, （341.19±63.99） μmol/L and （401.33±88.49） μmol/L, P< 0.05］. Compared with rigid plaque group (n=34), the serum UA level in soft plaque group (n=11) was significantly higher ［(389.00±69.45) μmol/L vs （323.03±72.71） μmol/L, P<0.05］. A stepwise multiple linear regression analysis demonstrated that age (r=0.414), systolic blood pressure (r=0.224), pulse pressure (r=0.270) and uric acid (r=0.219) were predisposed factors for higher CFPWV (P<0.05). CONCLUSION：UA is one of the risk factors causing hypertension. Serum UA level may reflect the severity and stability of carotid plaques. The increased arterial stiffness is closely related to the increased serum UA level in EH.     

Insulin-like growth factor I protects neonatal rat cardiomyocytes from apoptosis through improvement of mitochondrial function

AIM：To investigate whether mitochondrial mechanism is involved in the anti-apoptotic effect of insulin-like growth factor I (IGF-I) on cardiomyocytes. METHODS：Primary neonatal rat cardiomyocytes (NRCMs) were cultured and treated with 200 μmol/L hydrogen peroxide (H2O2) to induce apoptosis. Kruppel-like factor 9 (KLF9)-specific siRNA was transfected into the cells by Lipofectamine 2000. The mitochondrial function was measured by JC-1 mitochondrial membrane potential (MMP) assay. The mitochondrial morphology was observed by transmission electron microscopy. Myocardial cell apoptosis was detected by Annexin V-FITC/PI dual staining, caspase-3 activity assay, DNA-ladder analysis and Hoechst 33258 staining. RESULTS：The apoptosis of NRCMs was induced by H2O2, with MMP decreased by (24.0±1.6)% compared with control group. The fall rates of MMP in IGF-I group and KLF9 siRNA group were (18.3±1.2)% and (15.2±1.2)%, respectively (both P<0.01 vs H2O2 group), and improved mitochondrial morphology, decreased caspase-3 activity, attenuated DNA fragmentation and reduced apoptotic bodies were also observed in these two groups. The apoptotic rates of NRCMs in IGF-I group and KLF9 siRNA group were (22.4±4.2)% and (32.5±3.5)%, respectively, both lower than that in H2O2 group ［(42.5±1.8)%, P<0.01］. The anti-apoptotic effect of KLF9 silencing on NRCMs was consistent with that of IGF-I treatment. CONCLUSION：IGF-I protects NRCMs from apoptosis through down-regulating KLF9 expression and improving mitochondrial function.     

Complement activation in acute coronary syndromes

AIM: To evaluate complement activation in patients with all forms of acute coronary syndromes (ACS) and to examine the relationship between the degree of complement activation and myocardial injury.METHODS: The subjects were divided into 2 groups: 110 ACS patients (group ACS) and 18 healthy persons (group control).One hundred and ten patients with ACS were divided into 3 sub-group: 51 patients with ST-segment elevated myocardial infarction (STEMI),28 patients with non-ST-segment elevated myocardial infarction (NSTEMI) and 31 patients with unstable angina (UA).Complement 3 (C₃),complement 4 (C₄),troponin T (TnT) as well as creatine kinase MB (CK-MB) were evaluated.RESULTS: Plasma C₃ and C₄ peak levels were significantly higher in patients with STEMI ［(1 525±302)mg/L and (423±123) mg/L］ and NSTEMI ［(1 516±289)mg/L and (396±68) mg/L］ than those in patients with UA ［(1 275±172)mg/L and (356±91) mg/L］ and the control subjects ［(1 072±196)mg/L and (182±73) mg/L］ (P<0.01 for all).Also,C₃ and C₄ serum levels in patients with UA were significantly higher than those in control subjects (P<0.01 for all).At one-week follow-up,plasma levels of C₃ and C₄ were significantly different among various days in patients with ACS (P<0.01).Plasma C₃ and C₄ levels in ACS showed a relationship with peak creatine kinase MB (CK-MB) and troponin T (TnT) levels (P<0.01).CONCLUSION: Plasma C₃ and C₄ levels are elevated in ACS in present study.The relationship between C₃,C₄ levels and ACS suggests that the complement activation is related to necrosis within the myocardium.     

Inhibition of miR-9 expression suppresses proliferation,invasion and migration of nasopharyngeal carcinoma cells

AIM: To investigate the effects of down-regulated miR-9 expression on the proliferation, invasion and migration of nasopharyngeal carcinoma (NPC) cells. METHODS: Human NPC CNE1 and CNE2 cells were transfected with the inhibitor of miR-9 by Lipofectamine to down-regulate the expression of miR-9, and the cells transfected with an inhibitor control were also set up. The cell proliferation and cell cycle were evaluated by CCK-8 assay and flow cytometry. The cell invasion and migration abilities were detected by Transwell invasion and wound-healing assays. Immunoblotting was applied to analyze the levels of the proteins. RESULTS: Compared with control group, inhibition of miR-9 expression in the NPC cells by transfection of the miR-9 inhibitor significantly decreased the proliferation ability (P<0.05). The percentages of the cells in G0/G1 phase ［CNE2: (57.96±1.39)% vs (47.93±1.76)%, P<0.05; CNE1: (51.24±0.88)% vs (48.29±0.39)%, P<0.05］ were significantly increased. The migration distances ［CNE2: (186.50±7.94)μm vs (247.56±15.56)μm, P<0.05; CNE1: (139.06±16.73)μm vs (230.66±14.27)μm, P<0.01］ and the invasion ability of the CNE2 cells (43.00±3.17 vs 65.80±5.20, P<0.01) were also significantly inhibited. Moreover, the tumor cells transfected with the inhibitors produced lower β-catenin. CONCLUSION: Inhibition of miR-9 expression suppresses the proliferation, invasion and migration of nasopharyngeal carcinoma cells.     

Effects of beta2-adrenergic blocker on cytosolic Ca2+ in isolated cardiomyocytes of rats with myocardial infarction

AIM: To investigate if beta2-adrenergic receptors result in more Ca2+ load after myocardial infarction (MI), the effects of beta2-adrenergic blocker on cytosolic Ca2+ (［Ca2+］i) were studied. METHODS: Male Wistar rats underwent a ligation of left coronary artery (n=9) or a sham operation (n=3). Cardiomyocytes were dissociated at 2, 4 and 8 weeks after MI and ［Ca2+］i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol (1 μmol/L) in the presence or absence of atenolol (1 μmol/L), beta2-adrenergic blocker ICI118,551 (0.1 μmol/L) or propranolol (1 μmol/L) was examined. RESULTS: ICI118,551 suppressed the increase in ［Ca2+］i induced by isoproterenol at 4 and 8 weeks after MI (24.5%±5.7% vs 57.8%±13.2%, P<0.01; 12.2%±7.9% vs 44.6%±11.3%, P<0.01), but had no effects in control and 2 weeks post-MI groups. It decreased ［Ca2+］i in control and the three post-MI groups by 14.3%, 7.9%, 57.6% and 72.6%, respectively. Atenolol had suppressive effects only in control and 2 weeks post-MI groups (P<0.05). Propranolol had suppressive effects in control and all three post-MI groups (P<0.01). CONCLUSION: Beta 2-adrenergic blocker ICI118,551 exerts negative effects on ［Ca2+］i after MI, and the effects dramatically increase with the progression of MI.     

Effect of curcuma aromatica oil on learning,memory and hippocampal p-CaMKII expression in rats exposed to chronic hypoxia

AIM: To investigate the effects of different concentrations of curcuma aromatica oil on learning and memory in rats exposed to chronic hypoxia. METHODS: The rats were divided randomly into the control, chronic hypoxia and chronic hypoxia with low (LC), middle (MC) and high (HC) concentrations of curcuma aromatica oil groups. After 29 d, all animals were examined to obtain the scores of leaning and memory. The SOD activity and MDA content were determined in the serum and hippocampus, the ［Ca2+］_i in hippocampus was also detected. The staining and expression of p-calcium/calmodulin-dependent protein kinase II (p-CaMKII) in the hippocampus was observed and measured. RESULTS: ① In the chronic hypoxia group, the latency to find the hidden platform remarkably prolonged and the MDA content was obviously higher, but the SOD activity was significantly lower. Meanwhile, hippocampal ［Ca2+］_i was markedly increased. The immunostaining of p-CaMKII was much weaker in hippocampus as well as its expressions (P<0.01). ② The latency to find the hidden platform was remarkably shorter in groups with MC and HC (P<0.05). The MDA content was obviously lower among groups treated with curcuma aromatica, but SOD activity was significantly higher in groups with MC and HC. Meanwhile, hippocampal ［Ca2+］_i was markedly decreased in all groups treated with curcuma aromatica oil (P<0.01). The hippocampal immunostaining of p-CaMKII was much stronger in the MC and HC as well as its expression (P<0.05，P<0.01). Under the electron microscope, synaptic boundaries were not distinct, the edema of dendrite spine and axon was seen, synaptic vesicles and postsynaptic densities (PSD) were disappeared in the chronic hypoxia group. With rising of the concentration of curcuma aromatica oil, the edema of synapse and mitochondria was mitigated and the PSD was increased gradually. CONCLUSION: Curcuma aromatica oil might enhance learning and memory capacities of rats exposed to chronic hypoxia by cleaning up and antagonizing the production of the free radical and increasing the p-CaMKII expression in PSD. The effects are dose-dependent.     

Effects of adrenomedullin 2 on proliferation of microvascular endothelial cells from the rat cerebral cortex

AIM: To investigate the effects of adrenomedullin (ADM) 2 (AM2) on proliferation of microvascular endothelial cells from the rat cerebral cortex. METHODS: Microvascular endothelial cells (MEC) were isolated from the cerebral cortex of SD rats and cultured. The cultured cells were identified using immunocytochemistry assay with antibody for factor VIII-related antigen and randomly distributed into eight experimental groups as follows: control, AM2 10-7 mol/L, 10-8 mol/L, 10-9 mol/L, ADM, ADM+AM2, 10% fetal bovine serum (FBS) stimulated, and 10% FBS＋AM2 10^-7 mol/L groups. The proliferation of MEC was detected using ［3H］-TdR incorporation assay. RESULTS: Compared with control, AM2 (10^-7-10^-9 mol/L), ADM (10^-7 mol/L), and AM2 (10^-7mol/L) co-incubated with ADM (10^-7 mol/L) had no effects on ［3H］-TdR incorporation into the MEC (P>0.05). 10% FBS induced ［3H］-TdR incorporation increased by 87.5% (vs control, P<0.05), which was abolished by co-incubated the MEC with 10^-7 mol/L AM2 (P<0.05). CONCLUSION: AM2 inhibits FBS-stimulated proliferation of MEC from the rat cerebral cortex.     

Effect of renal sympathetic denervation on cardiac hypertrophy and fibrosis

AIM:To investigate the effect of renal sympathetic denervation (RDN) on cardiac hypertrophy and fibrosis and to explore the underlying mechanisms. METHODS:Male SD rats were randomly divided into 4 groups: sham, sham plus RDN, aortic constriction (AC) and AC plus RDN group (n=15 for each group). After the intervention for 8 weeks, the haemodynamic data and cardiac function were measured by a physiological recorder. The histological structure of the heart was evaluated by HE and picro-sirius red staining. The plasma concentration of norepinephrine (NE), renin activity, angiotensin II (Ang II) concentration and cardiac Ang II level were determined by radioimmunoassay. RESULTS:Compared with AC group, RDN improved cardiac diastolic function ［left ventricular end-diastolic pressure: (8.03±1.66) mmHg vs(15.77±2.14) mmHg; -dp/dt: (7 793±587) mmHg/s vs(6 353±475) mmHg/s; both P<0.01］, inhibited cardiac hypertrophy ［left ventricular index: 3.340±0.121 vs4.244±0.102; cardiomyocyte area: (332.9±28.9) μm2 vs(401.6±33.2) μm2; both P<0.01］ and attenuated cardiac fibrosis (collagen volume fraction: 7.76%±0.85% vs12.48%±1.82%; P<0.01) in aortic constricted rats. However, RDN didn’t cause significant reduction of blood pressure in aortic constricted rats (P>0.05). RDN prevented the AC-induced increase in the plasma NE concentration, renin activity, Ang II concentration and cardiac Ang II level. CONCLUSION: RDN may directly prevent cardiac hypertrophy and fibrosis and improve cardiac function via regulating the sympathomimetic activity and renin-angiotensin system.     

The clinical application of fluorescently-labeled monoclonal antibody against P-selectin

AIM: To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of P-selectin on platelet membrane is higher in DM (23.92%±15.83%), in hyperlipidemia (18.34%±9.46%) and in cerebral infarction (19.32%±10.38%) than normal subjects (3.38%±1.11%) (P<0.01). In addition, similar results on P-selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. CONCLUSION: FITC-labeled SZ-51-IgG can be used in FCM, and it would be a new and sensitive method in detecting platelet activation.     

Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.