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Objective To aims Study cardioprotection of different postconditioning algorithm,and investigate the role of MAPK pathway in the process.Methods Sixty SD rats were randomly (random number) divided into 5 groups,sham operation group,reperfusion/injury group (I/R group),gradual decreased reperfusion in postconditioning (GDR,30/10-25/15-15/25-10/30s of reperfusion/re occlusion) group,routine reperfusion in postconditioning (ER,4 cycles of 20/20s of reperfusion/reocclusion) group,and gradual increased reperfusion in postconditioning (GIR,10/30-15/25-25/15-30/10s of reperfusion/re-occlusion) group.Acute myocardial infarction and ischemic postconditioning models were established in the rats.Six hours after reperfusion,3 rats of each group were sacrificed and myocardial tissue were taken out to measure the level of phosphorylation of extracellular signal regulated protein kinase (P-ERK),Phosphorylation of stress-activated protein kinase (stress activated protein kinase,P-JNK),phosphorylation of p38 MAPK (P-p38),tumor necrosis factor-α (TNF-o),cysteine and aspartic protease-8 (Caspase-8) in myocardial tissue and the expression of cytochrome C in the cytosol using Western Blot method.Twenty-four hours after reperfusion all the remaining rats were to measure hemodynamic variables and level of myocardial enzyme,and myocardial tissue were taken out to determine myocardial apoptosis.A one-way analysis of variance (ANOVA) was used,and q tests were employed to determine differences in individual variables existed between groups.Results All three postconditioning modalities were found to provide cardioprotection (P ＜ 0.05) compared with I/R without preconditioning.The GIR provided the best cardioprotection effect,followed by ER and then GDR.Apoptotic index and serum marker levels were reduced far more in GIR than those in ER (P ＜ 0.05).The enhanced cardioprotection provided by GIR was accompanied by significantly increased levels of P-ERK1/2 [(1.82 ± 0.22) vs.(1.54±0.32),P＜0.05],and lower levels of P-p38 [(0.82±0.26) vs.(1.63 ± 0.24)],P-JNK [(0.76±0.28) vs.(1.33±0.21),TNF-o [(0.62±0.20) vs.(1.00±0.12)],Caspase-8 [(0.61 ±0.21) vs.(1.00±0.30)],Cyt-c [(0.66±0.16) vs.(1.68±0.22)] in the cytoplasm (P ＜ 0.05,all compared with ER).In addition,all the variables measured here were significantly improved in the GIR group relative to the GDR group (P ＜ 0.05).Conclusions The method of gradual increased reperfusion in postconditioning could attenuate reperfusion injury more significantly than routine algorithm whereby MAPK pathway played an important role in the process.
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AIM: To determine the effect of exogenous phosphocreatine (PCr) at different concentrations on transient outward potassium (Ito) current in rat ischemic ventricular mid-myocardial (M) cells and to explore the antiarrhythmia mechanism in the treatment of ischemic heart disease. METHODS: M cells were isolated enzymatically from left ventricular mid-myocardium of rats. Peak Ito current was recorded by patch-clamp technique in the whole-cell configuration when M cells were superfused with normal Tyrode solution,simple ischemic solution,and simulated ischemic solution containing PCr at concentrations of 5,10,20 and 30 mmol/L for 10 min. RESULTS: Peak Ito current density of M cells superfused with simple simulated ischemic solution was significantly reduced by (76.1±6.3)% (P0.05). CONCLUSION: PCr reverses the inhibition of Ito current under ischemic condition in M cells,which may be the mechanism responsible for arrhythmia prevention in ischemic heart disease. PCr at concentrations of 0～10 mmol/L exerts significant dose-effect relationship.
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BACKGROUND: Studies confirm that ischemia/reperfusion (I/R) injury can induce myocardial apoptosis. The loss of mitochondrial membrane potential (MMP) after reperfusion is the inevitable pathway of apoptosis. Protection of MMP may reduce apoptosis.OBJECTIVE: To observe the effect of naloxone on MMP of hypoxic myocardial cells and apoptosis in neonatal rats, and investigate the protective effect of naloxone on hypoxic myocardial cells.DESIGN: Observation and controlled trial.SETTING: Department of Cardiology, the General Hospital of Chinese PLA.MATERIALS: Detection of apoptosis of myocardial cells was carried out in the Laboratory of Pathophysiology, General Hospital of Chinese PLA in December 2004. Ten neonatal rats were used, and detection of MMP of myocardial cells was carried out in the same laboratory in March 2006 and 20 rats were used. All the involved rats were provided by the Animal Center of the General Hospital of Chinese PLA on the day of birth, were involved in this trial. Reagents: Naloxone hydrochloride injection (0.4 g/L, Beijing Sihuan Pharmaceutical Factory, Batch No. 0206272); the lowest and essential medium (Dulbecco, DEME,GIBCO company); phosphate buffer solution and fetal bovine serum (PBS and FBS, SIGMA Company).METHODS: The neonate rats were cut open along the median line of chest bone after local sterilization with iodine tincture on the day of birth. 1/3 ventricular myocardium before cardiac apex was harvested. On the 4th day of the culture,culture flasks of cells in good growth status ( ＞ 106 cells/bottle) were selected and divided into 3 groups: control group (normal culture, n =3 bottles), hypoxia group (hypoxia/reoxygenation, n =15 bottles) and naloxone group (hypoxia/reoxygenation, and treated by naloxone, n =15 bottles). Three time points were set in hypoxia group and naloxone group according to different time of hypoxia and reoxygenation: hypoxia 2 hours/reoxygenation 0 hour; hypoxia 2 hours/reoxygenation 2 hours; hypoxia 2 hours/reoxygenation 4 hours, 5 bottles at each time point. In the hypoxia group, DEME medium, which was pre-filled with 0.95 volume fraction of N2 and 0.05 volume fraction of CO2, containing 0.01 volume fraction of FBS, was used and 0.95 volume fraction of N2 and 0.05 volume fraction of CO2 was also filled to replace the air in the culture flasks. The culture flasks were enveloped for incubation at 37 ℃. The cells in the hypoxia group were incubated at normal condition (0.95 volume fraction of air and 0.05 volume fraction of CO2) at set time. In the naxolone group, hypoxia/reoxygenation treatment was the same as above, and naloxone hydrochloride was added at the sametime and the final concentration of naloxone in culture flasks was 5 μmol/L (The volume for naloxone ≤ 0.5% of the total medium). In the control group, hypoxia/reoxygenation and naloxone treatment were not given, but the same volume of normal saline was added, and this time served as time point of hypoxia 0 hour/reoxygenation 0 hour. After intervention,myocardial MMP changes and apoptosis were detected with fluorescent staining-flow cytometer at different time after hypoxia/reoxygenation.MAIN OUTCOME MEASURES: ① MMP changes of hypoxia group and naloxone group at different time points; ② Comparison of survival, apoptosis and necrosis of cells at hypoxia 2 hours/reoxygenation 4 hours in each group.RESULTS: ① After hypoxia, MMPs of the cells in hypoxia group and naloxone group were decreased. MMP of the cells in the naloxone group was higher than that in the hypoxia group at each time point (P ＜ 0.01). The great amplitude of decrease of MMP occurred in hypoxia period, but not in reoxygenation period. ② At hypoxia 2 hours/reoxygenation 4 hours, the apoptotic and necrotic rates in the hypoxia group were significantly higher than those in the naloxone group [(9.88±0.98)% vs. (2.41±0.52)%; (5.10±0.29)% vs. (3.56±0.56)%, both P ＜ 0.01]. The apoptotic rate was significantly higher than the necrotic rate in the hypoxia group (P ＜ 0.05).CONCLUSION: Early application of naloxone can significantly alleviate and postpone the decrease of myocardial MMP after I/R, and reduce apoptosis and necrosis.
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BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ADMSCs) have the multilineage differentiation potential, and are relatively easier to be obtained, thus they have attracted more and more attention as a new seed cell for cell engineering.OBJECTIVE: To observe the in vitro culture conditions of ADMSCs isolated from rat's subcutaneous adipose tissue, and identify them using immunohistochemical staining.DESIGN: An animal experiment.SETTING; Department of Cardiology, the General Hospital of Chinese PLA.MATERIALS: One healthy male Wistar rat of clean degree, 4 months old, weighing 200 g, was used. DMEM, fetal bovine serum were from GIBCO; Monoclone antibodies of rabbit-anti-rat CD13, CD34, CD44, CD45, CD105, D-related human leucocyte antigen (HLA-DR), factor-Ⅷ, vov Willebrand factor (VWF), Myosin, SABC kits and DAB staining kit from Wuhan Booster Biological Engineering, Co.,Ltd; Adeno-associated virus encoding green fluorescent protein from Vector Gene Technology Company Limited (Beijing).METHODS: The experiments were carried out in the Department of Internal Medicine, the General Hospital of Chinese PLA in October 2006. ① Cell isolation and culture: 0.3 g adipose tissue was cut from subcutaneous adipose tissue of Wistar rat's groin under aseptic condition, then minced and digested before culture, DMEM was changed at 2-3 days after plenty of fusiform-shap ed attached cells were observed under microscope, and the cell growth was observed. The cell concentration was adjusted to 2×107 L-1, then seeded into 96-well plate, and 100 μL for each well. From the second day, 3 wells were randomly selected every day, the cells were released with tripsin, and counted with blood cell counting chamber under inverted microscope. ② Cell viability assay: ADMSCs of passages 3 to 8 were added to DMSO freeze medium, and thawed after 2-4 weeks, and the cell viability was assessed by trypan blue dye exclusion. ③Immunohistochemical staining and identification: 2 ×107 L -1 cells were seeded to culture plate, then the immunohistochemical (SABC method) identification and Oil red O staining were performed to determine the cell surface antibodies of CD13, CD34, CD44, CD45, CD105, HLA-DR, factor-Ⅷ, HLA-DR and VWF. ④Lineage-specific differentiation and identification: The ADMSCs were plated on multi-well chamber and induced with lineage-specific media supplementation at least two weeks and identified by histologic/immunohistochemical assay of Oil red O for adipogenisis, alkaline phosphatase (ALP) stain for osteogenisis and Myosin monoclonal antibody for myogenisis. ⑤Transfected adenovirus carried green fluorescence protein (AD-GFP) medium: The fourth generation of ADMSCs were seeded on 96-well plate, 3 000 cells for each well, serum-free DMEM was changed after 24 hours, and added by AD-GFP at the same time, then transfected with different multiplicity of infection (MOI) of 1∶50, 1∶100, 1∶150 and 1∶200respectively, and then the transfection was observed.MAIN OUTCOME MEASURES: ① Results of cell isolation and culture; ② Cell viability after freezing and thawing; ③Results of immunohistochemical staining and identification; ④ Results of lineage-specific differentiation and identification;⑤ Results of transfected adenovirus carried AD-GFP.RESULTS: ① About 3.6×105 attached cells were obtained from 0.3 g subcutaneous adipose tissue, and these cells could be subcultured for passages in vitro with stable population doubling time. ② The cells were thawed after freezing for 2-3 weeks, and the trypan blue staining showed that the cell viability was above 90%. ③ The immunocytochemical staining showed that CD13, CD44, CD105 were positive and CD45, factor-Ⅷ, HLA-DR and VWF negative in different generations. ④ From the second generation, a few Oil red O positively stained cells were observed, which were obviously increased after prolonging the refreshing. After lineage-specific differentiation, the cells were all positive by Oil red O staining, ALP staining and Myosin immunohistochemical staining. ⑤ 72 hours after transfection, it was observed under fluorescence microscope that most cells were green fluorescence when the MOI value was 1∶200, the transfection was successful, and it was generally determined that the transfection rate was above 90%.CONCLUSION: A large number of ADMSCs with multilineage differentiation potential can be easily obtained from rat adipose tissue, osteoblast, myoblasts, they can be expanded in large quantity and stored in vitro for long time, AD-GFP were also successfully transfected.
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To explore the changes of mitochondrial membrane potential in rat cadiomyocyte apoptosis by fluorescent JC 1, H 2 O 2 was used to induce cadiomyocyte apoptosis, and JC 1 in combination with flow cyometry was used to detect the changes of mitochondrial membrane potential in the early stage of cadiomyocyte apoptosis. The results showed that living cardiomyocytes had a high mitochondrial membrane potential, JC 1 aggregates were formed in the inner membrane of mitochondria and emitted orange red fluorescence. H 2 O 2 caused the decrease of mitochondrial membrane potential, JC 1 aggregates were dissociated to monomer,which emitted green fluorenscence. So the red fluorescence decreased. It is suggested that JC 1 in combination with flow cyometry is an ideal method to detect the changes of mitochondrial membrane potential.
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Prostaglandin E1 ( PGE1) , derived from D-HLA and controlled by phospholipidase A2, is a kind of strong endogeneous vasodilator and platelet inhibitor. Its effects on vasculars and platelets are just inferior to prostacyclin only, which is the strongest physiological vasodilator and platelet inhibitor, and the common receptors exits.Now it is found that exogeneous PGE1 can central some kinds of paletet supra-activation in illnesses such as cardiovascular disease, renal disease and diabetes mellitus. So PGE1 may be benificial to those patients.
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Objective To compare and assess the antihypertentive efficacy and safety of barnidipine hydrochloride and felodipine in the elderly hypertentive patients. Methods 69 elderly patients(60-75 years old ) with mild or moderate essential hypertension were selected and randomly assigned to two groups, one was medicated with barnidipine (36 patients) and another (33 patients) with felodipine. Placeboes were given for 2 weeks, then the testees were administered barnidipine 10mg/d or felodipine 5mg/d for 2 weeks, followed by an increased administration of dose titration to barnidipine 15mg/d or felodipine 10mg/d for another 2 weeks in those patients with DBP≥90mmHg. Both groups were administered once daily. Results After 4 weeks administration no statistical difference was found on the total effectiveness between barnidipine and felodipine groups (100.0% vs 93.9%). Both SBP and DBP declined by 20.7?7.8mmHg and 17.6?4.5mmHg respectively in barnidipine group, and 20.2?9.6mmHg and 17.6?6.1mmHg respectively in felodipine group. The decline of blood pressure showed no significant difference between the two groups. The mild harmful respond occurred in 4 of 36 patients of barnidipine group (11.1%), and in 3 of 33 patients of felodipine group(9.1%).Conclusion The results suggested that barnidipine is effective and well tolerated in antihypertension of the old patients.
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On the basis of superimposed stenosis, a model of femoral occlusive thrombus was produced in 12 dogs. Among them, urokinase+saline were given in 6 dogs (group A), aptamer was given ten minutes before urokinase in another 6(group B). The aim of the study was to explore the effect of a new direct sited thrombin inhibitor thrombin aptamer on thrombolysis . The results showed that the interval between thrombosis and reflow was significantly different between this two groups ( P
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DynaPulse, a newly developed noninvasive apparatus, is valuable in assessing blood pressure, cardiac output and vascular compliance. The purpose of the present study was to evaluate the validity of cardiac output measurement using DynaPulse in patients, compared with echocardiography and Fick method. Forty five inpatients underwent cardiac output measurement using DynaPulse (CO DP ) and Doppler echocardiography (CO DE ), cardiac output was measured with DynaPulse (CO DP ) and Fick method (CO FM ) in additional 26 patients. A good correlation was found between CO DP and CO DE ( r =0 76). There was an acceptable correlation between CO DP and CO FM ( r =0 61). DynaPulse can provide a noninvasive, clinically useful estimation of cardiac output.