Protection of Glycyl-L-Glutamine against myocardial ischemia-reperfusion injury in the isolated rat heart

AIM: To study the protection of Glycyl-L-Glutamine(Gly-Gln) against myocardial ischemia/reperfusion(I/R) injury in the isolated rat heart.METHODS: A model of myocardial ischemia-reperfusion injury was established with a Langendorff apparatus. Thirty male SD rats were randomly divided into four groups: control group, Gly-Gln group, I/R group and I/R+Gly-Gln group. Both I/R and I/R+Gly-Gln group were pre-perfused for 30 min, followed by 20 min ischemia and 40 min reperfusion. During reperfusion I/R+Gly-Gln group was perfused with Gly-Gln perfusate. Control group was kept perfused for 90 min. Gly-Gln group Gly-Gln perfusate was also kept perfused for 90 min. The left ventricular end-diastolic pressure(LVEDP), left ventricular developed pressure (LVDP), ±dp/dtmax, heart rate (HR), monophasic action potentials(MAP) was measured during perfusion. The coronary effluent fluid was collected at different certain times. The activities of lactic dehydrogenase (LDH) and creatine kinase(CK) were determined.RESULTS: The isolated rat heart function decreased severely after 20 min ischemia and 40 min reperfusion(I/R): the LVEDP increased and the LVDP, ±dp/dtmax decreased. But the LVEDP decreased and the LVDP, ±dp/dtmax increased in I/R+Gly-Gln group compared with I/R group. Moreover, the activities of LDH and CK in the coronary effluent fluid decreased remarkably in I/R+Gly-Gln group compared with I/R group.CONCLUSION: Gly-Gln can play a protective role against myocardial I/R injury in isolated rat hearts via maintaining the left ventricular function and decreasing the release of LDH and CK.     

Effects of human urotensin II on ischemia/reperfusion injury in isolated perfused rat hearts

AIM: To investigate the effects of human urotensin II (hUII) on ischemia/reperfusion (I/R) injury in isolated rat hearts. METHODS: In the ischemia/reperfusion (I/R) model of isolated perfused rat hearts, the effects of hUII pretreatment on cardiac function was monitored with cardiac function software of MFL Lab200. ATP, total calcium, and malondialdehyde (MDA) content in myocardium were detected. The coronary perfusion flow (CPF) and lactate dehydrogenase (LDH) activity in coronary effluent were measured during reperfusion. RESULTS: In the hUII pretreated group, the release of LDH from myocardium was lower [(78.3±18.1)U/L] than I/R group [(109.3±23.9) U/L, P< 0.05], with decreased contents of MDA and calcium in myocardium (decreased by 24% and 27%, respectively, P< 0.05) and an increased myocardial ATP content [(3.8±0.4)μmol/g dw vs (2.2±0.4)μmol/g dw, P< 0.05)]. At the same time, hUII pretreatment increased CPF [(5.4±0.7) mL/min vs (3.8±0.8) mL/min in I/R group, P< 0.05], reduced left ventricular end-diastolic pressure (LVEDP) by 20% ( P< 0.05) with increased±d p /d t max [(217±38) kPa/s and (119±18) kPa/s vs (173±29) kPa/s and (82±25) kPa/s in I/R groups, respectively, P< 0.05]. hUII pretreatment also increased natrite/natrate (NO₂^-/NO₃^-) content in coronary effluent [(52.2±12.0)μmol/L vs (32.1±10.2)μmol/L in I/R group, P< 0.05)]. CONCLUSION: hUII pretreatment attenuated I/R injury in isolated perfused rat hearts. The protective mechanism might be associated with NO-mediated coronary vasodilation.     

Effects of diabetes on ischemic/reperfusion injury and cell apoptosis in rats myocardium via alterations in peroxynitrite

AIM: To determine the effects of different-term streptozotocin(STZ)-induced diabetes on ischemia/reperfusion (I/R) injury and cell apoptosis in rats myocardial via alterations in myocardial peroxynitrite.METHODS: The models of I/R injury were induced by occlusion and reperfusion of the left descending coronary artery (LDCA) in rats.I/R-induced infarct size was determined using triphenyltetrazolium chloride (TTC) staining.Quantified caspase-3 expression was used to represent apoptosis by Western blotting analysis.Peroxynitrite formation as indicated by nitrotyrosine level was measured by morphometric analysis.RESULTS: Two weeks after STZ treatment,infarct size (35.00%±3.00%) was smaller in 2 weeks diabetic hearts (2WKD) as compared with time-matched control group (2WKC) (51.00%±3.30%),whereas after 16 weeks of diabetes (16WKD),the infarct size (61.00%±3.00%) was bigger in the diabetic hearts as compared with the 16WKC group (50.00%±2.00%,P<0.05).After I/R,caspase-3 expression was lower in 2WKD+I/R group (A value：481±77) than that in 2WKC+I/R group (A value：1033±46),while caspase-3 was higher in the 16WKD+I/R group (A value：1206±78) than that in 16WKC+I/R group (A value：940±72,P<0.05).Nitrotyrosine was lower in 2WKD hearts (A value：211±13) than that in controls (A value：409±12),but was higher in 16WKD group (A value：506±37) compared with controls (A value：378±46,P<0.05).CONCLUSION: Short- and long-term STZ induced diabetes exerts opposite influences on myocardial I/R injury and cell apoptosis,and these contradictory influences may depend on different alterations in myocardial peroxynitrite.     

Ischemic postconditioning reduces myocardial damage in rats suffering from limb ischemia-reperfusion

AIM: To investigate the protective effect of limb ischemic postconditioning on the myocardial damage in the rats suffering from limb ischemia-reperfusion (LIR). METHODS: Wistar rats were randomly divided into control group (C group), ischemia-reperfusion group (IR group) and ischemic post-conditioning group (IR+IPostC group). For conducting ischemic postconditioning, the rats in IR+IPostC group underwent 5 min of ischemia and 5 min of reperfusion on their hind limbs repeatedly after 4 h of ischemia, and then, 4 h of reperfusion was applied. The activity of superoxide dismutase (SOD), xanthine oxidase (XOD) and myeloperoxidase (MPO) was measured. The levels of malonaldehyde (MDA) in plasma and myocardial tissues, the levels of creatine kinase (CK), creatine kinase MB (CK-MB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (α-HBDH) and myocardial troponin I (cTnI) were also detected. The changes of ultrastructure in the myocardium were observed under electron microscope. RESULTS: Compared with C group,the levels of CK-MB, AST, LDH,α-HBDH and cTnI were all increased in IR and IR+IPostC groups. The levels of MDA and XOD also increased (P<0.05), but the activity of SOD decreased (P<0.05). However, compared with group IR, the levels of CK-MB, AST, LDH, α-HBDH and cTnI decreased (P<0.05) in IR+IPostC group.The levels of MDA and XOD also decreased (P<0.05), but the activity of SOD increased (P<0.05). Under electron microscope, the cardiac myofibrils arranged neatly, light and dark bands were clear, the mitochondrial cristae arranged closely and neatly, and the mitochondrial matrix densification was observed in C group. However, the cardiac fiber arrangement was disordered or disappeared, stromal edema was obvious, most or all mitochondrial cristae and membrane became fusion or disappeared, mitochondrial vacuolization and decrease in glycogen were obvious in IR group. In IR+IPostC group, the pathological changes mentioned above were attenuated somewhat than those in IR group. CONCLUSION: Ischemic postconditioning protects rat myocardium under limb ischemia-reperfusion.     

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.     

Liver X receptors attenuate myocardial ischemia-reperfusion injury through modulating GLUT-4

AIM：To investigate whether liver X receptors (LXRs) attenuate myocardial ischemia-reperfusion (I/R) injury in isolated rat heart through modulating glucose transporter 4 (GLUT-4). METHODS：Isolated rat hearts were used to establish the model of ischemia-reperfusion injury using Langendorff apparatus. The hearts were divided into 7 groups: LXR agonist T0901317 (0.1, 0.5 and 10 μmol/L) pretreatment groups, ischemic preconditioning group, control group, control+DMSO group, and I/R group. The releases of lactate dehydrogenase (LDH) and creatine kinase (CK), the infarct size, the hemodynamic parameters (left ventricle developed pressure,left ventricle end-diastolic pressure, coronary flow and ±dp/dt max), the relative mRNA level of GLUT-4 and the protein content of GLUT-4 in the myocardial cell membrane were compared between these groups. RESULTS：Besides producing hemodynamic disorders, I/R increased the activities of LDH and CK, and the infarct size in model groups. Treatment with T0901317 significantly suppressed ischemia-reperfusion injury-induced increases in LDH and CK, and reduced the infarct size. T0901317 also significantly ameliorated the parameters of haemodynamics. Treatment with T0901317 significantly increased GLUT-4 expression at mRNA and protein levels in the myocardial cell membrane. CONCLUSION：Liver X receptors may attenuate myocardial ischemia-reperfusion injury in isolated rat heart by modulating the expression of GLUT-4.     

Inhibitory effect of ischemic postconditioning on autophagy induced by focal cerebral ischemia reperfusion in rats

AIM: To investigate the effect of ischemic postconditioning (IPC) on autophagy induced by focal cerebral ischemia reperfusion (I/R) in rats. METHODS: Healthy male SD rats were assigned randomly into sham-operation (sham) group, I/R group and IPC group with 10 rats in each group. The rats in sham group were only exposed the right common, internal and external carotid artery surgically. The rats in I/R group were subjected to right middle cerebral artery occlusion (MCAO) by the modified Longa suture method for 2 h followed by 24 h of reperfusion. The rats in IPC group were subjected to MCAO for 2 h followed by reperfusion of the ipsilateral common carotid artery occlusion for 10 s for 5 episodes, and then reperfusion for 24 h. Autophagy was obeserved by transmission electron microscopy (TEM). The protein levels of mammalian target of rapamycin (mTOR), p-mTOR and microtubule associated protein light chain 3 (LC3)-II in brain tissue of the rats were determined by Western blot. Pathological changes of brain tissue were observed by HE staining. RESULTS: The protein levels of mTOR and p-mTOR in IPC group were significantly higher than those in I/R group (P<0.05). The expression of LC3-II in IPC group was significantly lower than that in I/R group (P<0.01). The cerebral infarction area and brain water content in IPC group were significantly lower than those in I/R group (P<0.01). HE staining showed that neurons degeneration and necrosis in IPC group were significantly alleviated compared with I/R group. TEM observation showed that IPC revealed fewer autophagosomes, with much less severe cell damage than that in I/R group. CONCLUSION: IPC reduces brain ischemia reperfusion damage by decreasing autophagy of brain cells, which might be related to the activation of mTOR.     

Ischemic postconditioning protects skeletal muscle from ischemia/reperfusion injury through improving microcirculation in rat hind limb

AIM: To investigate the effect of ischemic postconditioning (I-postC) on ischemia/reperfusion (I/R) injury of skeletal muscle and its microvasculatory mechanisms in rat hind limb. METHODS: Healthy male Wistar rats were randomly divided into three groups as follows (n=16): I/R group, I-postC group and ischemic preconditioning (IPC) group. I/R of right hind limb was induced by clamping right femoral artery. The effects of I-postC on oxygen saturation of hemoglobin (StO2), blood flow, microcirculation, hemodynamics, Wet/Dry ratio (W/D) and ultramicrostructure of skeletal muscle were detected on 12 h or 24 h after reperfusion. RESULTS: (1) Compared with I/R group, 12 h after reperfusion, W/D from I-postC group decreased 6.7% (P<0.05) and myofibrillae lysis of skeletal muscle and vacuolation of mitochondria attenuated than that in I/R group. 24 h after reperfusion,-dp/dtmax in I-postC group increased 10.46% (P<0.05). (2) It was also found that 24 h after reperfusion, blood velocity in venule increased 11.3% in I-postC group compared with that in I/R group (P<0.05), and internal diameter of arteriole and venule increased 39.8% and 24.1% compared with that in I/R group, respectively (P<0.01). No significant difference between I-postC and IPC groups (P>0.05) was found. CONCLUSION: I-postC protects the skeletal muscle against reperfusion injury by improving microcirculation.     

Effects of Panax quinquefolium saponin on mitochondrial membrane potential and cardiomyocyte apoptosis in ischemia/reperfusion rats

AIM：To investigate whether mitochondrial membrane potential (ΔΨm) and the mitochondrial apoptotic pathway are involved in the protective mechanism of Panax quinquefolium saponin (PQS) against cardiomyocyte apoptosis after ischemia/reperfusion (I/R) injury in rat myocardium. METHODS：Ninety healthy male SD rats were randomly divided into sham group, I/R group, PQS (200 mg·kg-1·d-1) +I/R group, cyclosporine A (CsA) group, CsA (10 mg·kg-1) +I/R group and PQS +CsA +I/R group. The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion in the rats. The serum activity of lactate dehydrogenase (LDH) was measured by automatic chemistry analyzer. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosolic cytochrome C were determined by Western blotting. ΔΨm was measured by laser scanning confocal microscopy and fluorescence microplate reader. RESULTS：Compared with I/R group, the serum content of LDH,the infarction size in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group and the myocardial apoptotic index were decreased. Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) of ΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group. Moreover, in those 3 groups, the protein levels of cleaved-caspase-3 and cytosolic cytochrome C were decreased compared to I/R group, respectively. CONCLUSION：PQS attenuates myocardial injury and cardiomyocyte apoptosis during I/R, and the protective mechanisms of PQS were associated with the modulation of ΔΨm and the inhibition of mitochondrial apoptosis pathway.     

Effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion-induced early liver injury in rats

AIM：To explore the effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion (IR)-induced early liver injury. METHODS：Adult SD rats (n=35) were randomized into 5 groups with 7 rats each: sham operation group (S group), IR group, cromolyn sodium treatment group (IR+C group, 25 mg/kg), ketotifen treatment group (IR+K group, 1 mg/kg), compound 48/80 treatment group (IR+CP group, 0.75 mg/kg). IR was induced by superior mesenteric artery occlusion for 75 min followed by 4 h of reperfusion. The agents were intravenously administered 5 min before reperfusion. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and histamine, and the liver levels of lactate dehydrogenase (LDH), tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed. The liver histopathologic changes were also evaluated. RESULTS：IR resulted in severe liver injury as demonstrated by great increases in injury scores, concomitant significant increases in serum levels of AST, ALT and histamine, and liver levels of LDH, TNF-α, IL-8, and MDA, accompanied by reduced SOD activity (all P<0.05 vs S group). Treatment with cromolyn sodium or ketotifen markedly alleviated IR-mediated liver injury as confirmed by significant reduction of the above biomedical changes, whereas compound 48/80 further aggravated liver injury by dramatically enhancing the biomedical changes (all P<0.05 vs IR group). CONCLUSION：Inhibition of mast cell function before reperfusion may reduce early liver injury induced by intestinal ischemia reperfusion. Histamine, oxidative stress and inflammatory response may provide promising effects on it.     

Effects of ischemic preconditioning on oxidative stress and mitochondrial function in young and old rat myocardium with ischemia/reperfusion

AIM: To study the protective effect of ischemia preconditioning (IPC) on ischemia/reperfusion (IR)-damaged myocardium in young and old rats. METHODS: Male Wistar rats aged at 3 months (young) and 20 months (old) were used to establish myocardial IPC model and IR model with the method of Langendorff heart perfusion. The rats were divided into young ischemia/reperfusion (YIR) group, young ischemic preconditioning (YPC) group, old ischemia/reperfusion (OIR) group and old ischemic preconditioning (OPC) group. Transmission electron microscopy was used to observe the ultrastructural changes of myocardial tissue and myocardial mitochondria. The myocardial infarction area was determined by TTC staining. The lactate dehydrogenase (LDH) content in coronary effluent fluid and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in myocardial tissues were detected by the method of colorimetry. The levels of nitrated and carbonylated proteins in myocardial tissue were measured by ELISA. The myocardial cell apoptosis was analyzed by TUNEL assay. The mitochondrial respiratory function and mitochondrial permeability transition pore opening induced by calcium load were evaluated by oxygen electrode method. RESULTS: Compared with YIR group, the myocardial infarction area in YPC group was obviously smaller, SOD activity in myocardial tissues increased, LDH activity in coronary effluent fluid and the content of MDA decreased, and the levels of nitrated and carbonylated proteins in the cardiac tissues reduced. In YPC group, the mitochondrial membrane structure appeared intact, cristae of the mitochondria showed close arrangement, and the matrix was compressed under the electron microscope. Myocardial mitochondrial respiratory control rate, state Ⅲ oxygen consumption and the P/O ratio in YIR group all significantly increased, proton leak decreased, mitochondrial swelling induced by calcium distinctly reduced, and myocardial apoptosis rate declined. No significant difference of the above indexes between OIR group and OPC group was observed. Compared with YPC group, myocardial ultrastructural damage increased clearly, cardiac oxidative stress increased, mitochondrial respiratory function declined, and cell apoptosis and necrosis increased in OPC group. CONCLUSION: Ischemic preconditioning has protective effect against myocardial IR injury in young rat hearts, while old rat hearts were less sensitive to ischemic preconditioning, leading to bluntness of cardioprotection with IPC in aging hearts. This may be related to mitochondrial injury and severe cellular apoptosis caused by increase of cardiac oxidative stress levels in the aging ischemic preconditioning heart.     

Effects of simvastatin on expression of Bcl-2 and Bax in myocardium of rats with renal ischemia-reperfusion injury

AIM: To observe the effect of simvastatin on myocardial tissue after renal ischemia-reperfusion injury and its mechanism. METHODS: A rat model of renal ischemia-reperfusion injury was prepared by clamping the bilateral renal arteries for 45 min. The rats (n=36) were randomly divided into sham operation group, renal ischemia-reperfusion (I/R) group and simvastatin group with 12 rats in each group. The content of serum creatinine (SCr), blood urea nitrogen (BUN) and myocardial tissue malondialdehyde (MDA), the myocardial activity of lactate dehydrogenase (LDH), creatine kinase (CK) and superoxide dismutase (SOD), and the myocardial protein expression of Bcl-2 and Bax were detected. RESULTS: Compared with sham operation group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in I/R group were significantly increased (P<0.05), and the activity of SOD was significantly decreased (P<0.05). Compared with I/R group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in simvastatin group were significantly decreased (P<0.05), while SOD activity was enhanced (P<0.05). The protein expression of Bcl-2 and Bax in sham operation group was less than that in I/R group (P<0.05), and the protein level of Bax in simvastatin group was significantly lower than that in I/R group (P<0.05), while the protein level of Bcl-2 was increased (P<0.05). CONCLUSION: Simvastatin has a protective effect on the myocardium of the rats with renal ischemia-reperfusion injury, and the protective mechanism may be related to the elimination of free radicals by simvastatin, increase in the protein expression of Bcl-2 and decrease in the protein expression of Bax.     

Effect of diazoxide postconditioning on cardiac function and mitochondrial cardiolipin in isolated rat heart

AIM: To investigate the effect of diazoxide (D) postconditioning on Cardiac function and mitochondrial cardiolipin in isolated rat heart and to explore the protective effect of ATP sensitive potassium channel on diazo-xide postconditioning myocardium. METHODS: The myocardial ischemia/reperfusion injury model in isolated rat hearts was established by Langendorff apparatus. The isolated rat hearts were randomized into 4 groups (n=8): control group (control), myocardial ischemia/reperfusion injury group (I/R), diazoxide postconditioning group (I/R+D), 5- hydroxy decanoic acid (5-HD) plus diazoxide postconditioning group (I/R+5-HD+D). The hearts in each group were started with 20 min perfusion for equilibration. The hearts in control group perfused for 70 min; The hearts in I/R group was global ischemia for 40 min after ischemia reperfusion at 4 ℃ ST. Thomas cardioplegia, then reperfusion for 30 min; The hearts in I/R+D group were treated with diazoxide (50 μmol/L) in K-H perfusion for 5 min after global ischemia for 40 min, then reperfusion for 25 min; The hearts in I/R+5-HD+D group were treated with 5-HD (100 μmol/L) in K-H perfusion for 5 min before diazoxide postconditioning, then reperfusion for 20 min. The heart rate, coronary outflow volume, heart function, myocardial enzymes and myocardial mitochondrial cardiolipin at the end of perfusion in each group were determined. RESULTS: Compared with control group and I/R+D group, the heart rate, the concentration of heart phospholipid and the coronary outflow volume were reduced, the heart function was significantly impaired the contents of myocardial enzymes were increased in I/R group. However, no significant difference between I/R group and I/R+5-HD+D group was observed. CONCLUSION: The diazoxide postconditioning protects the myocardium by increasing mitochondrial cardiolipin content, reducing the release of myocardial enzymes, improving heart function and reducing myocardial reperfusion injury. The myocardial protective effect of diazoxide is completely blocked by 5- hydroxy decanoic acid.     

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.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Role of cAMP signaling in cardioprotective mechanism of ischemic postconditioning

AIM: To assess the role of the cAMP signaling in cardioprotection by brief intermittent ischemia at the time of onset of reperfusion (i.e. postconditioning). METHODS: The model of rat myocardial ischemia/reperfusion (I/R) was used. The left ventricular functions were assessed by measuring the left ventricular developed pressure (LVDP) and the coronary flow (CF). The injury of myocardium was further confirmed by detecting the releases of lactate dehydrogenase(LDH) and creatine kinase(CK) in coronary effluent. The mRNA expression of caspase-3, bcl-2 and bax in myocardium was determined by real-time PCR. RESULTS: I/R treatment led to the decrease in LVDP and CF, and the increase in the releases of CK and LDH in coronary artery effluent. The mRNA expression of caspase-3 and bax/bcl-2 ratio was up-regulated simultaneously. Postconditioning treatment relieved the injury induced by I/R, which was enhanced by the specific phosphodiesterase 4(PDE4) inhibitor rolipram. On the other hand, the specific adenylyl cyclase inhibitor SQ22536 attenuated those protective effects of postconditioning. CONCLUSION: The cAMP signaling participates in the protective effect of postconditioning on heart from I/R injury, and the effect may be associated with the regulation of apoptosis.     

Effects of ethane 1,2-dimethanesulfonate preconditioning on renal ischemia/reperfusion injury in male rats

AIM: To investigate the effects of ethane 1,2-dimethanesulfonate (EDS) preconditioning on renal ischemia/reperfusion (I/R) injury in male Sprague-Dawley (SD) rats. METHODS: Male SD rats (n=48) were randomly assigned to 6 groups: blank, sham, I/R, EDS+I/R, EDS+testosterone (TST)+I/R, and castration (Cast)+I/R. The renal pedicles were bilaterally occluded with a microvascular clamp for 45 min to establish renal I/R-induced injury model. Bilateral orchiectomy was conducted 2 weeks before surgery. EDS (75 mg/kg) was intraperitoneally injected 5 d before operation. Blood samples were collected 24 h after reperfusion from the vena orbitalis posterior plexus. Luteinizing hormone (LH), TST, serum creatinine (SCr), blood urea nitrogen (BUN), and kidney injury molecule-1 (KIM-1) were detected. The renal tissues were harvested to measure the level of TNF-α and the expression of Fas mRNA and caspase-3 protein. RESULTS: Serum TST levels in EDS+I/R group and Cast+I/R group were below the minimum detectable threshold. Compared with other groups, the rats in EDS+I/R group and Cast+I/R group had higher levels of SCr, BUN and KIM-1 (P<0.05). SCr and BUN levels showed no significant difference between EDS+I/R group and Cast+I/R group (P>0.05), but KIM-1 level in EDS+I/R group was lower than that in Cast+I/R group (P<0.05). After reperfusion for 24 h, the levels of TST and LH in EDS+I/R group, Cast+I/R group and EDS+TST+I/R group were lower than those 1 h before operation (P<0.05). Compared with Cast+I/R and I/R group, the TNF-α level and expression of Fas mRNA and caspase-3 protein were significantly decreased in EDS+I/R group (P<0.05). CONCLUSION: EDS preconditioning substantially reduces the serum TST level, thus attenuating I/R-induced acute renal injury. TNF-α-induced Fas/FasL pathway may be involved in this process.     

Effects of diosmin on activity of myeloperoxidase in renal tissues and expression of CD11b,CD54 and CD62L on neutrophils in rats with kidney ischemia and reperfusion

AIM：To observe the effects of diosmin on the activity of myeloperoxidase (MPO) in renal tissues and the expression of CD11b, CD54 and CD62L on neutrophils in rats with kidney ischemia and reperfusion. METHODS：One hundred and eighty Sprague-Dawley rats were randomly divided into 3 groups: sham operation (SO) group, ischemia and reperfusion (I/R) group and diosmin+I/R (DOSM+I/R) group. At the end of the experiment, renal tissues were obtained and the activity of MPO was detected by ELISA. The expression of CD11b, CD54 and CD62L on polymorphonuclear leukocytes was determined by flow cytometry after monoclonal antibody labeling. RESULTS：The renal MPO activity at different time points in DOSM+I/R group was significantly lower than that in I/R group (P<0.01 or P<0.05). The expression levels of CD11b, CD54 and CD62L on polymorphonuclear leukocytes in I/R group were significantly higher than those in SO group (P<0.01 or P<0.05), while those in DOSM+I/R group were lower than those in I/R group (P<001 or P<0.05). CONCLUSION: Diosmin not only decreases the activity of MPO in renal tissues, but also reduces the expression of CD11b, CD54 and CD62L on polymorphonuclear leukocytes, suggesting that its protective effect against kidney ischemia-reperfusion injury through anti-inflammatory mechanism.     

The protection of the hepatic ischemic preconditioning is concerned with the NO/ET-1 system

AIM: To study the relationship between the disturbance of nitric oxide/endothelin-1(NO/ET-1) and hepatic ischemia/reperfusion(I/R) injury as well as the regulation of NO/ET-1 system by hepatic ischemic preconditioning(IPC). METHODS: The changes of NO/ET-1 system and their relationship with hepatic I/R injury were compared between I/R group and IPC+I/R group in a rat hepatic I/R model. Two hours after reperfusion, the liver tissues were detected by RT-PCR to see whether there was inducible nitric oxide synthase (iNOS) mRNA expression. RESULTS:In the acute phase of hepatic reperfusion, the ratio of NO/ET-1 was reduced, which was due to a significant reduction of NO₂^-/NO₃^- (the metabolic product of NO) and significant elevation of ET-1 in the blood plasma. The content of ALT, AST, LDH and TNF-α in blood plasma, and of MDA in liver tissue were increased but ATP in liver tissue was reduced, the hepatic damage was deteriorated. The protection of the hepatic IPC was concerned with the elevation of the ratio of NO/ET-1 caused by the elevation of NO₂^-/NO₃^-, and reduction of ET-1 as well. There was no iNOS mRNA detected in the liver tissues.CONCLUSION: Hepatic I/R injury is related to the disturbance of NO/ET-1. The protection of the hepatic IPC in the acute phase might be conducted by its regulation of NO/ET-1 system. The cNOS rather than the iNOS generated the NO in this situation.     

X-ray irradiation increases sensitivity of myocardium to ischemia/reperfusion injury in rats

AIM：To observe the sensitivity of myocardium to ischemia/reperfusion (I/R) injury in the rats with chest radiotherapy. METHODS：The radiation-induced heart disease model was established by local 20 Gy of X-ray irradiation in the chest. Male Wistar rats (n=42) were randomly divided into 6 groups:sham trauma group, trauma group, sham trauma+sham operation group, sham trauma +I/R group, trauma+sham operation group and trauma+I/R group. The rats were subjected to 30 min of ischemia and 1 h of reperfusion 2 week after trauma. The left ventricular developed pressure (LVDP) and ±dp/dtmax were recorded by BL-410 biological signal recording and analysis system. The serum cardiac troponin I (cTnI) and creatine kinase isoenzyme (CK-MB) were measured by ELISA. The myocardial infarct size was determined by nitroblue tetrazolium(NBT) staining method and BI2000 image analysis software. RESULTS：Compared with sham trauma+I/R group, significant decreases in LVDP and ±dp/dtmax were observed in trauma+I/R group (P<0.01) with significant increases in the infarct size and the concentrations of cTnI and CK-MB (P<0.01). CONCLUSION:Chest X-ray irradiation increases the sensitivity of myocardium to I/R injury in rats.