Role of apelin in rat myocardial ischemic preconditioning and ischemia/ reperfusion injury

AIM: To study the alteration and role of apelin in myocardial ischemic preconditioning and ischemia/reperfusion injury of rats.METHODS: Forty-five SD rats were randomly divided into three groups: ischemia/reperfusion group (IR),ischemia pre-conditioning group (IP) and sham operation group.ECG was continuously used to evaluate the score of arrhythmias.The protein levels of apelin-36 in myocardium and plasma were detected by radioimmunoassay.The expression of apelin was observed by immunohistochemistry.RESULTS: (1) The scores of arrhythmias in IP group (2.1±0.5) was only 58.3% of IR group (3.6±0.8) ( P<0.01).(2) The apelin-36 protein level of plasma and myocardium in IR group were respectively lower by 36.1% and 45.6% than those in SH group (P<0.01),and those in IP group were lower by 23.8% and 24.7% than those in SH group (P<0.01),but higher than those in IR group (18.9% and 38.5%,respectively,P<0.05).(3) The staining absorbance of apelin in IR,SH and IP group was (7.87±2.41),(22.53±2.54) and (14.23±2.15),respectively.There were significant differences between IR and SH (P<0.01) and between IP group and SH group (P<0.05).(4) The scores of arrhythmias in IP and IR were negatively correlated with the protein level of apelin-36 in myocardium (r= 0.847,P <0.01).CONCLUSION: The down-regulation of apelin-36 in the plasma and myocardium of rats indicates that apelin has an important role in myocardial ischemic preconditioning and ischemia/reperfusion injury.     

Effect of HSP70 on HBV replication

AIM: To investigate the role of heat shock protein 70(HSP70)in hepatitis B virus (HBV) replication. METHODS: The effect of HBV replication on the expression of HSP70 was analyzed by RT-qPCR. The overexpression efficiency of HSP70 was confirmed by Western blot. The effect of HSP70 overexpression on HBV DNA replicative intermediates was analyzed by RT-qPCR and Southern blot. The effects of HSP70 overexpression on the expression level of HBV 3.5 kb mRNA and HBV core protein were measured by RT-qPCR and Western blot, respectively. The Effect of HSP70 overexpression on HBV promoter activity was detected by dual luciferase reporter system. RESULTS: The mRNA levels of HSP70 were inhibited by HBV replication. Overexpression of HSP70 repressed the expression of HBV DNA replicative intermediates, 3.5 kb mRNA and core protein, as well as HBV core promoter activity. CONCLUSION: HBV replication inhibits the expression of HSP70. Overexpression of HSP70 represses HBV replication. These data suggest that HSP70 repressed HBV replication by inhibiting HBV core promoter activity.     

Effect of PKC activition on cardiac myocyte apoptosis and Bcl- 2 expression during myocardial ischemia/reperfusion in rats

AIM: To explore the effect of PKC activition on cardiac myocyte apoptosis and expression of bcl- 2 during myocardial ischemia/reperfusion(I/R) in rats. METHODS: TUNEL,immunohistochemistry and in situ hybridization were used. RESULTS: The TUNEL data showed that the numbers of positive cardiac myocyte nucleus and the percentage of positive cardiac myocyte nucleus in PMA+IR_{3 h} group decreased significantly(P＜0.05,P＜0.01), compared to those in IR_3h group. The number of Bcl-2 protein positive cardiomyocytes and the percentage of Bcl-2 protein positive cardiomyocytes in PMA+IR_3h group were higher than those in IR_3h group (P＜0.01) bcl- 2 mRNA expression showed the same changes in PMA+IR_0h group compared to IR_1h group.CONCL USIONS:Activation of PKC decreased cardiomyocyte death during I/R.Upregulation of bcl-2 gene expression in cardiomyocytes during I/R may be one of the mechanisms of decreasing cardiomyocyte death by PCK activating during I/R.     

Effect of cGMP-dependent protein kinase on endothelial cytoskeleton changes

AIM: To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of burn shock. METHODS: Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with 10% burn serum or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 were used to pretreat the endothelial cells before the administration of burn serum or PKG activator 8-Br-cGMP. RESULTS: Exposures to burn serum and 8-Br-cGMP led to a rapid time-dependent increase in endothelial PKG activity and the polar distribution of intracellular filamentous actin, and preincubation with KT5823 abolished those effects. CONCLUSIONS: The results suggest that burn serum induces PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably contributes to the endothelial hyperpermeability after burn shock.     

Effects of HSP90 on complement-mediated hypoxia/reoxygenation injury of H9c2 cardiomyocytes during hypoxic postconditioning

AIM To investigate the effects and mechanisms of heat shock protein 90 （HSP90） on complement-mediated hypoxia/reoxygenation （H/R） injury of rat H9c2 cardiomyocytes during hypoxic postconditioning （HPC）. METHODS Rat H9c2 cardiomyocytes were divided into 7 groups according to different treatments： （1） control group （cultured for 10 h under normal oxygen）; （2） H/R group （hypoxia for 4 h and reoxygenation for 6 h）; （3） HPC group （3 cycles of 5 min H/R after hypoxia for 4 h, followed by reoxygenation for 6 h）; （4） HPC+geldanamycin （GA） group （1 μmol/L HSP90 inhibitor GA was added 20 min before HPC）; （5） negative control group （empty plasmid was transfected before HPC）; （6） C3 over-expression group （C3a plasmid was transfected before HPC）; （7） C5 over-expression group （C5a plasmid was transfected before HPC）. Morphological changes of the H9c2 cells were detected by Hoechst 33242 staining. The effects of HPC on the apoptosis of H9c2 cells were examined by flow cytometry. The protein levels of HSP90, C3a, C5a, NF-κB p65, TNF-α, IL-1β, IL-6, Bcl-2 and Bax were determined by Western blot. RESULTS With up-regulation of HSP90, HPC significantly reduced H/R-induced apoptosis of the H9c2 cells, inhibited the expression of C3a, C5a, NF-κB p65, TNF-α, IL-1β, IL-6 and Bax, and increased the expression of Bcl-2. These effects were blocked by GA. The inhibitory effects of HPC on NF-κB p65 expression and H9c2 cell apoptosis were offset after over-expression of C3a or C5a. CONCLUSION HSP90 attenuates H/R injury of H9c2 cardiomyocytes by inhibiting complement-NF-κB signaling pathway during HPC.     

Effects of apelin-13 on transient sodium currents in rat ischemic ventricular myocardial cells

AIM：To investigate the effects of apelin on ventricular arrythmias and cardiac functions in rat Langendorff perfusion-simulated myocardial ischemia model by observing the changes of transient sodium currents (INa) in normal cells and the simulated ischemic cells in rat left ventricle. METHODS：Ventricular cells were enzymatically isolated by the Langendorff perfusion system. INa was recorded by the technique of whole-cell patch-clamp. Some elements in the extracellular fluid were changed to simulate the normal or ischemic status. Forty Wistar rats were divided into 4 groups：normal group, ischemic group, normal with apelin group and ischemic with apelin group. The effect of apelin-13 on INa was observed. The method of rat Langendorff perfusion was used to simulate the ischemic heart model. The ventricular arrhythmia scores and heart functional parameters were compared. The expression level of sodium channel protein,type V,alpha subunit (SCN5A) in ventricular ischemic cells was measured by Western blotting. RESULTS： Apelin-13 increased INa amplitude in both normal myocardial cells ［(-86±13） pA/pF］ and ischemic myocardial cells ［(-52±15） pA/pF］. The results of current-voltage curve analysis indicated that apelin-13 did not change the conduction velocity of INa in the 4 groups ［(3.2±0.2） pS/pF, （3.1±0.3） pS/pF,（2.9±0.1）pS/pF and （2.8±0.4） pS/pF,respectively, P>0.05］. The membrane potentials at 50% maximal activation in the 4 groups were （-21.9±0.6） mV, （-28.7±0.3） mV, （-30.5±0.7） mV and （-36.8±0.2） mV, respectively, and the slope of activation curves was 5.6±0.3, 5.1±0.4, 4.3±0.3 and 4.9±0.6 (P>0.05), respectively. No difference of ventricular arrhythmia scores between normal group and normal with apelin group, as well as between ischemic group and ischemic with apelin group was observed. LVEDP in normal with apelin group was lower than that in normal group.The dp/dtmax and dp/dtmin in normal with apelin group were higher than those in normal group. Apelin improved cardiac function parameters in the ischemic hearts. The expression of SCN5A was not affected by apelin (28.8±3.6, 29.4±4.1, 30.1±2.9 and 31.3±3.8,respectively,P>0.05). CONCLUSION：Apelin-13 changes the gating properties of sodium channel, enhances the peak INa and facilitates the opening of sodium channel without inducing ventricular arrhythmias. Apelin-13 has a positive inotropic effect on both normal and ischemic hearts.     

Role of heme oxygenase-1 in the ischemic preconditioning of isolated rat heart

AIM: To investigate the influence of ischemic preconditioning on heart function, the activities of lactate dehydrogenase (LDH), malondialdehyde (MDA), and heme oxygenase-1 (HO-1) after ischemia/reperfusion in isolated rat heart. METHODS: The model of Langendorff was used in isolated rat heart perfusion. Ischemic preconditioning protocol: stopping perfusion for 5 minutes and reperfusion for 5 minutes, repeating three times. Ischemia protocol: stopping perfusion for 40 minutes and reperfusion for 20 minutes. Indexes of heart function were recorded in control M8, ischemia and reperfusion group (IR), and ischemic preconditioning group (IPC). The content of LDH of coronary effluent was measured. Moreover, the content of MDA and activity of HO-1 in myocardium were also measured. RESULTS: The recovery percentage of heart function in IPC group was significantly higher than that in IR group (P<0.01) and the activity of heme oxygenase-1 also increased significantly (P<0.05). CONCLUSION: The contents of LDH and MDA significantly decreased in IPC group compared with IR group. The increase in heme oxygenase-1 activity might be involved in the protective effect of ischemic preconditioning on ischemic/reperfused rat heart.     

Effects of ischemic preconditioning on cardiac myocyte apoptosis and expression of bcl-2 during myocardial ischemia/reperfusion in rats

AIM:To explore the effect of ischemic preconditioning on cardiac myocyte apoptosis and the expression of bcl-2 during myocardial ischemia/reperfusion in rats. METHODS:We use TUNEL,immunohistochemical and in situ hybridization(ISH) methods to detect the cardiac myocyte apoptosis and the expression of bcl-2 during myocardial ischemia/reperfusion in rats. RESULTS:①The numbers of positive cardiac myocyte nuclear and the percentage of positive cardiac myocyte nuclear in IP+I/R_3h group decreased significantly(P<0.05,P<0.01)compared with I/R_3h group,respectively.②The numbers of bcl-2 protein positive cardiomyocyte and the percentage of bcl-2 protein positive cardiomyocyte in IP+I/R_3h group were higher(P<0.01)than that of I/R_3h group,respectively.The numbers of positive bcl-2 mRNA cardiomyocyte and the percentage of positive bcl-2 mRNA cardiomyocyte in IP+I/R_1h group were higher(P<0.01)than that of I/R_1h group,respectively.CONCLUSION:① The first window of IP's protection could reduce cardiomyocyte apoptosis significantly.② Up-regulating the protein expression of bcl-2 in cardiomyocytes during I/R may be one of the mechanisms of first window of IP's protection.     

Effects of norepinephrine preconditioning and ischemic preconditioning on apoptosis and Bcl-2, Bax expression in rat myocardial cells during myocardial ischemic reperfusion

AIM: To study the effects of norepinephrine preconditioning(NE-P) and ischemic preconditioning (IP)on apoptosis and Bcl-2, Bax expression in rat myocardial cells in myocardial ischemic reperfusion (I/R). METHODS: The model of rat ischemic-reperfusion was used to conduct NE-preconditioning. Apoptotic myocytes were detected with TUNEL. Bcl-2, Bax expression were detected with immunohistochemistry. RESULTS: The rate of apoptosis cells in I/R group was higher, the rate of apoptosis cells in NE-P group and IP was lower significantly than that in I/R group(P＜0.01). The expression of Bcl-2 in I/R group was lower, but the expression of Bax was higher, the expression of Bcl-2 in NE-P group was higher significantly than that in I/R group(P＜0.01), the expression of Bax in NE-P group was lower than that in I/R group(P＜0.01). There was no significantly difference between NE-P and IP group in the above parameters (P＞0.05). CONCLUSION: NE-P reduced myocyte apoptosis by I/R in rats; The expression of Bcl-2 ,Bax genes played an important role in myocardial apoptosis.     

Effect of adenosine infusion before ischemic preconditioning on immature myocardial reperfusion injury in neonatal rabbits

AIM and METHODS:To investigate the cardioprotective effect of adenosine infusion before ischemic preconditioning on immature myocardial reperfusion injury in rabbit heart. Isolated perfused working heart model were performed, all hearts were subjected to 2-hour global hypothermic ischemia and received intermittent cold cardioplegia perfusion.RESULTS:During reperfusion, the recovery of left ventricular systolic pressure, left ventricular end-diastolic pressure, +dp/dt_max, and -dp/dt_max of hearts received adenosine infusion before ischemic preconditioning were significantly improved, myocardial adenosine triphosphate and adenosine diphosphate content and superoxide dismutase activity were higher, the leakage of myocardial creatine kinase and the malondialdehyde content were lower, and myocardial water content was obviously less.CONCLUSION:These results suggest adenosine infusion before ischemic preconditioning enhances cardioprotection of ischemic preconditioning against immature myocardial reperfusion injury in the rabbit heart.     

Cardiac protective effect of granulocyte colony-stimulating factor combined with ischemic postconditioning on acute myocardial infarction in rabbits

AIM: To investigate the protective effect of granulocyte colony-stimulating factor (G-CSF) combined with ischemic postconditioning (IP) on acute myocardial infarction (AMI). METHODS: Male New Zealand rabbits were randomly divided into 4 groups (n=15) after 30 min of left ventricular artery (LVA) occlusion: the rabbits in ischemia-reperfusion (IR) group were directly given reperfusion|the rabbits in G-CSF group were subsequently treated with G-CSF (10 μg·kg^-1·d^-1) by subcutaneous injection after direct reperfusion|the rabbits in IP group received 4 episodes of 30 s reperfusion and 30 s occlusion before total reperfusion|the rabbits in IP combined with G-CSF (IP+G-CSF) group were treated with both IP and G-CSF. Electrocardiogram (ECG) monitoring was performed during the operation. Blood was drawn to evaluate white blood cell count (WBC) and cardiac troponin I (cTnI) before operation and 7 d later. Ultrasound cardiography was performed to evaluate left ventricular remodeling and functions 4 weeks after operation. The sizes of infarcted myocardium were determined by triphenyltetrazolium chloride (TTC) staining. Apoptosis of cardiomyocytes was measured by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: ST-segment resolutions were significantly decreased in IP group and IP+G-CSF group compared with direct reperfusion groups (P<0.05). WBC significantly increased in the groups treated with G-CSF for 1 week. The values of cTnI after operation were significantly lowered in G-CSF group, IP group and IP+G-CSF group as compared with IR group (P<0.05). Left ventricular ejection fraction, the size of infarcted myocardium and apoptosis of cardiomyocytes were better in IP group, G-CSF group and IP+G-CSF group than those in IR group. CONCLUSION: G-CSF combined with IP is a promising strategy against cardiac reperfusion injury and accelerates cardiac repair in AMI.     

Effect of ginsenoside Rg1 on activity and protein expression of NOS in rats with cerebral ischemic reperfusion injury

AIM: To observe the neuroprotective effects of ginsenoside Rg1 on focal cerebral ischemia reperfusion (I/R) injury in rats. METHODS: SD rats were applied to right middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. The rats were randomly divided into sham-operation group, I/R group and ginsenoside Rg1 pretreatment groups. The rats in ginsenoside Rg1 pretreatment groups were pretreated with ginsenoside Rg1 at doses of 10, 20 or 40 mg/kg once a day for 7 days and then subject to MCAO. The neurological deficit score was measured by Longa's method. The neurons were observed with Nissel staining. The nitric oxide (NO) content, the activity of nitric oxide synthase (NOS) and inducible NOS (iNOS) in the brain tissues were determined. The expression of neuronal NOS(nNOS) and iNOS was detected by Western blotting. RESULTS: Compared with sham-operation group, ginsenoside Rg1 significantly reduced the neurological deficit score and increased the neuron number in the hippocampus. The activity of NOS and iNOS, and NO content were decreased. Ginsenoside Rg1 also down-regulated the expression of nNOS and iNOS. CONCLUSION: Ginsenoside Rg1 has protective effect on the brain during cerebral I/R injury in rats. The mechanism may be related to reducing the content of NO and the activiy of NOS dose-dependently.     

Effect of heat shock protein on dendritic cell maturation

AIM: To study the effect of heat shock protein (HSP) on the maturation of dendritic cells (DCs) and observe the morphological changes of DCs dynamically. METHODS: The HSP (gp96)-peptide complex was purified from the tissues of hepatocellular carcinoma. Hepatoma cells were treated by heat shock for preparation of the HSP expressed on cell surface and then marked with DiI fluorescence. Immature DCs from peripheral blood mononuclear cells were cocultured with two types of HSPs. The morphological changes of DCs were observed dynamically and the effects of HSPs on the maturation of DCs analyzed by Flowcytometer. RESULTS: The morphological changes and the processes of antigen capture of DCs cocultured with DiI marked tumor cells were well showed. Four ways to capture antigens of DCs were observed, including direct contact, besieging, forming bubbles and extending pseudopodia with bubbles on the terminals. Results also indicated that both types of HSP could promote the maturation of DCs. CONCLUSION: DiI is a good fluorescent dye suitable for the morphological studies of DCs. Four ways for DCs to capture antigens were indicated in this paper. Different types of HSP, purifed from tumor tissues or expressed on tumor cells surface, promote DC maturation, and the purified HSP is more effective.     

Nitric oxide opens the second window of protection in ischemic preconditioning via induction of heat shock protein 72

AIM:To examine the inhibition of nitric oxide (NO) synthesis during ischemic preconditioning (IP) on the induction of heat shock protein 72 (HSP72) and infarct size-limiting effect of the second window of protection. METHODS:Rabbits were subjected to 4 cycles of 5 min of coronary artery occlusion separated by 10 min reperfusion, or received a sham operation. During this procedure, N^G-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) was injected intravenously 5 min before IP followed by its continuous infusion. Twenty-four hours later, the hearts were rapidly excised for assaying HSP72 expression or were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion and then measured infarct size (IS). RESULTS:Twenty-four hours later, immunoblotting revealed an increase in HSP72 protein levels in the IP group, and this was blocked by L-NAME. IS of the IP rabbits was reduced as compared with the control (29.8%±3.7% vs 50.8%±4.3%, P＜0.01). IS in the IP rabbits was elevtated as a result of L-NAME treatment (46.0%±5.1%). Administration of L-arginine reversed the effects of L-NAME on the induction of HSP72 and IS (33.5%±4.0%). The intravenous administration of S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) increased the induction of HSP72 and reduced IS (31.3%±5.7%, P＜0.01vs control) 24 h later. CONCLUSION:These findings suggest that NO may be involved in the induction of HSP72 and the opening of the second window of protection of IP.     

Effect of ischemic preconditioning on myocardial Bcl-2 expression and mitochondrial structure during heart valve replacement surgery

AIM: To investigate the effect of ischemic preconditioning (IP) on myocardial Bcl-2 expression and mitochondrial structure during heart valve replacement surgery under cardiopulmonary bypass. METHODS: Fifty-four patients were prospectively randomized to receive or not ischemic preconditioning (IP) before cold cardioplegic arrest. Ischemic preconditioning in the IP patients (n=22) was induced by a single 2-min ischemia followed by 3-min reperfusion just before aortic clamping and cold crystalloid cardioplegia for myocardial protection. The control group (n=32) received no ischemic preconditioning before cold cardioplegic arrest. The levels of ejection fraction (EF), fractional shortening(FS) and stroke volume (SV) in both groups were measured and compared. troponin T (c-TnT) level, Bcl-2 protein expression and microscopic changes of myocardial mitochondrial structure were recorded for each group before and after surgery. RESULTS: The level of EF, FS and SV in IP group was higher than those in control group (P<0.05). No significant difference in preoperative c-TnT levels between two groups was observed. The level of c-TnT in IP group was lower than that in control group and with a declining trend over time of 6 h, 24 h, 48 h, 72 h and 5 d after surgery, respectively. The preoperative positive unit of Bcl-2 expression between two groups showed no statistical difference (P> 0.05). Postoperatively, the positive unit of Bcl-2 expression in IP group was 19.85±5.88, significantly increased as compared to the preoperative value (P<0.05). In control group, the positive unit of Bcl-2 expression was 14.17±3.39, showed no statistically significant difference to the preoperative value (P>0.05). Postoperative Bcl-2 expression between two groups showed a significant difference (P<0.05). In the control group, microscopic observation revealed swollen mitochondrion, with a hardly visible or disrupted membrane for some mitochondrion; mitochondrial crista were obviously dissolved and loose with a large number of vacuoles formation. However in IP group, myocardial mitochondrion appeared with intact membrane, concentrated mitochondrial cristae with high electron density and no vacuoles formation was observed. CONCLUSION: IP may up-regulate the expression of myocardial anti-apoptotic protein Bcl-2 to protect the mitochondrion, thus protecting cardiocytes and cardiac functions.     

Effect of zhiganning on the expressions of HSP60 and HSP70 in the hepatocytes of rats undergoing steatohepatitis

AIM: To observe the expressions of HPS60 and HPS70 in hepatocytes in rats under treatment with zhiganning on steatohepatitis. METHODS: Male SD rats were randomly divided into large dose zhiganning group, small dose zhiganning group, ursodeoxycholic acid (UDCA) group, model group, normal control group. Except the normal control group, all the other rats were fed with high fat (88% standard diet, 10% lard, 2% cholesterol) and 35% alcohol 10 mL/kg twice a day. Prophylactic drugs were used at the same time. All rats were sacrificed at the 9th week. Routine histologic features of hepatic sections were observed by HE staining and penetrated electron microscope. The expressions of HSP60 and HSP70 in the liver were detected by immunohistochemistry, respectively. RESULTS: ⑴ The degree of steatohepatitis in the large dose zhiganning group and ursodeoxycholic acid (UDCA) group were significantly decreased compared with that in model group (P<0.05). ⑵ The expression of HSP70 in the large dose zhiganning group and ursodeoxycholic acid (UDCA) group were significantly higher than that in either model group or normal control group (P<0.05, P<0.01, respectively). The expression of HSP60 in the large dose zhiganning group was significantly higher than that in either model group or normal control group (P<0.05, P<0.01, respectively). ⑶ In the large dose zhiganning group and ursodeoxycholic acid group, ultramicroscopic structure of liver was nearly normal, which was significantly improved compared with model group. CONCLUSION: The results indicate that zhiganning and UDCA effectively prevente the steatohepatitis in rats induced by high fat diet and alcohol. The enhanced expressions of HSP60 and HSP70 may play an important role in the prevention of liver from injury.     

Effect of protein kinase C signal pathway on resistin expression in 3T3-L1 adipocytes

AIM：To investigate the effect of protein kinase C on resistin expression in 3T3-L1 adipocytes.METHODS：The differentiated 3T3-L1 adipocytes were incubated with 50 nmol/L phorbol 12-myristate 13-acetate (PMA) or 5 μmol/L Ro-31-8220 for 24 h.Expression of resistin mRNA was detected by RT-PCR and expression of resistin protein was detected by Western blotting.RESULTS：Compared with control,PMA increased the expression of resistin mRNA and protein in 3T3-L1 adipocytes significantly (P<0.01),while Ro-31-8220 decreased the expression of resistin mRNA and protein in 3T3-L1 adipocytes obviously (P<0.01).CONCLUSION：Protein kinase C signal pathway may regulate resistin expression in 3T3-L1 adipocytes.     

Effects of spermine on myocardial ischemia/ reperfusion injury in rats

AIM: To observe the effect of exogenous spermine (low concentration) on myocardial ischemia/reperfusion injury in rats.METHODS: 40 Wistar rats were randomly divided into 4 groups: sham- operation group (Sham), ischemic reperfusion group (I/R), spermine group (Sp) and natural saline group (NS). The model of ischemic/reperfusion injury was established by ligating rat coronary artery. In Sp group, spermine (0.5 mmol/L, 2 mL/kg) was injected slowly into rat vein. During the process, we recorded the electrocardiogram and the LV functional parameters, assayed the levels of SOD, LDH, NO and MDA in serum, and examined the ultrastructure of the myocardium. RESULTS: In I/R group, the incidence of arrhythmia was 90%, myocardial ultrastructure was injured seriously, values of LVSP and ±dp/dtmax decreased, levels of LDH, NO and MDA increased while SOD activity decreased (P<0.05 or P<0.01, compared with Sham group). Compared with I/R and NS group, all those indexes in Sp group changed significantly (P<0.05 or P<0.01). CONCLUSION: Exogenous spermine alleviates myocardial ischemia/ reperfusion injury in rats. The mechanism may be related to its antioxidant effect and relieving the injury caused by oxygen free radical.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Effects of ligustrazine ferulate on myocardial apoptosis and apoptosis-related protein expression in rats with myocardial ischemia-reperfusion injury

AIM:To observe the effects of ligustrazine ferulate on the apoptosis of myocardial cells in rats with myocardial ischemia-reperfusion injury, and to explore its possible mechanism. METHODS:Sixty male SD rats were randomly divided into five groups: sham-operation group, ischemia-reperfusion group, ligustrazine (4 mg/kg) group, low-dose (4 mg/kg) ligustrazine ferulate group and high-dose (8 mg/kg) ligustrazine ferulate group. The rat myocardial ischemia-reperfusion model was established by 30 min of myocardial ischemia followed by 120 min of reperfusion. Drugs were administered to the rats by jugular vein injection 10 min before reperfusion. After the reperfusion was finished, the biochemical indicators in serum and the histological indexes in myocardium were detected. RESULTS: Compared with ischemia-reperfusion group, ligustrazine ferulate lowered the serum levels of creatine kinase MB form, lactate dehydrogenase, cardiac troponin I and malondialdehyde, elevated the activity of total superoxide dismutase in serum and the expression of Bcl-2 protein in myocardium, decreased the expression of Bax protein and myocardial apoptotic index, and increased the Bcl-2/Bax ratio (all P<0.01). All the indicators in ligustrazine ferulate groups were dose-dependently superior to those in ligustrazine group (P<0.05 or P<0.01). CONCLUSION: Ligustrazine ferulate protects rats against myocardial ischemia-reperfusion injury. Its anti-apoptotic effect may be related to up-regulation of Bcl-2 and down-regulation of Bax.